Method for the preparation of 2,4-dihydroxybutyrate

ABSTRACT

A method for the preparation of 2,4-dihydroxybutyric acid from homoserine includes a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and a second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate.

This is a Continuation of application Ser. No. 14/414,331 filed Jan. 12,2015, which in turn is a National Stage Application of PCT/EP2013/064619filed Jul. 10, 2013, which claims the benefit of U.S. ProvisionalApplication No. 61/670,405 filed Jul. 11, 2012. The disclosure of theprior applications is hereby incorporated by reference herein in theirentireties.

The present invention relates to a novel method for the preparation of2,4-dihydroxybutyrate (2,4-DHB) from homoserine comprising a two-steppathway:

-   -   a first step of conversion of the primary amino group of        homoserine to a carbonyl group to obtain        2-oxo-4-hydroxybutyrate, and    -   a second step of reduction of the obtained        2-oxo-4-hydroxybutyrate (OHB) to obtain 2,4-DHB.

The carboxylic acids cited within the present application are equallynamed under their salt (e.g. 2,4-dihydroxyburyrate) or acid forms (e.g.2,4-dihydroxybutyric acid).

2,4-dihydroxybutyric acid (equally 2,4-DHB or DHB) is a compound ofconsiderable economic interest. DHB can be readily converted intoα-hydroxy-γ-butyrolactone in aqueous media by adjusting the appropriatepH. α-hydroxy-γ-butyrolactone is a prominent precursor for theproduction of the methionine substitute2-hydroxy-4-(methylthio)-butyrate (HMTB) (US 2009/318715) which has alarge market in animal nutrition. At present, α-hydroxy-γ-butyrolactoneis derived from γ-butryolactone by a multi-stage process that implieshalogenation of the γ-butryolactone in position α, and subsequentsubstitution of the halogen atom by a hydroxyl group in alkaline medium(US 2009/318715).

From growing oil prices the need for the production of DHB fromrenewable resources arises. Microorganisms are capable of transformingbiomass-derived raw material, e.g. sugars or organic acids, into a largevariety of different chemical compounds (Werpy & Petersen, 2004). Withthe growing body of biochemical and genomic information it is possibleto modify microorganisms such that they overproduce naturally occurringmetabolic intermediates with high yield and productivity (Bailey, 1991).Optimization of production microorganisms often requires rationalengineering of metabolic networks which ensures, among others,overexpression of enzymes required for the biosynthesis of themetabolite of interest, and alleviation of product feedback inhibition.Another possibility is the implementation of novel enzymatic systemsthat catalyze the production of a metabolite of interest.

Metabolic engineering approaches and enzymatic catalyses requiredetailed knowledge of the biochemistry and regulation of the metabolicpathway leading to the metabolite of interest. In the case of DHBproduction, this information is not available. Only few studies reportthe occurrence of DHB in patients with succinic semialdehydedehydrogenase deficiency (Shinka et al., 2002) without, however,identifying enzymatic reactions implicated in DHB production. Thezymotic or enzymatic production of DHB, therefore, requires (i) theidentification of a thermodynamically feasible pathway which transformsan accessible precursor into DHB, (ii) the identification orconstruction of enzymes that are capable to catalyze individual reactionsteps in the pathway and (iii) the functional expression of the pathwayenzymes in an appropriate production organism. The present invention hasas an objective to satisfy these needs.

Accordingly, one object of the present invention is a method ofpreparation of 2,4-DHB from homoserine comprising a two-step pathway(see FIG. 1):

-   -   a first step of conversion of the primary amino group of        homoserine to a carbonyl group to obtain OHB, and    -   a second step of reduction of the obtained OHB to 2,4-DHB.

The first and/or the second step(s) of the method of the invention canbe catalyzed either by an enzyme encoded by an endogenous or aheterologous gene.

In the description, enzymatic activities are also designated byreference to the genes coding for the enzymes having such activity. Theuse of the denomination of the genes is not limited to a specificorganism, but covers all the corresponding genes and proteins in otherorganisms (e.g. microorganisms, functional analogues, functionalvariants and functional fragments thereof as long as they retain theenzymatic activity).

Within a further aspect of the invention, the enzyme converting theprimary amino group of homoserine to a carbonyl group to obtain OHB canbe homoserine transaminase, homoserine dehydrogenase, or homoserineoxidase.

Within a further aspect of the invention, the enzyme having homoserinetransaminase activity can be identified among enzymes having aspartatetransaminase (EC2.6.1.1) activity, branched-chain-amino-acidtransaminase (EC2.6.1.42) activity, or aromatic-amino-acid transaminase(EC2.6.1.57) activity.

Within a further aspect of the invention, the homoserine transaminasecan be the branched-chain-amino-acid transaminase from Escherichia coli,Ec-IlvE, and Lactococcus lactis, Ll-BcaT, the aromatic-amino-acidtransaminases from E. coli, Ec-TyrB, L. lactis, Ll-AraT, andSaccharomyces cerevisiae, Sc-Aro8, or the aspartate transaminase from E.coli, Ec-AspC.

The second step of the method of the present invention is catalysed byan enzyme having OHB reductase activity. Within a further aspect of theinvention, the enzyme having OHB reductase activity can be identifiedamong enzymes having 2-hydroxyacid dehydrogenase activity, in particularamong enzymes having lactate dehydrogenase (Ldh) (EC1.1.1.27,EC1.1.1.28), malate dehydrogenase (Mdh) (EC1.1.1.37, EC1.1.1.82,EC1.1.1.299) activity, or branched chain (D)-2-hydroxyacid dehydrogenase(EC1.1.1.272, EC1.1.1.345) activity. More specifically, the enzymehaving homoserine transaminase activity is encoded by genes ilvE, tyrB,aspC, araT, bcaT, or ARO8.

In an even more specific aspect, the enzyme having homoserinetransaminase activity is encoded by sequence set forth in SEQ ID NO: 59,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 or SEQ ID NO:69 or any sequence sharing a homology of at least 50% with saidsequences or corresponds to SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 or any sequence sharing ahomology of at least 50% with said sequences.

Within a further aspect of the invention, the OHB reductase enzyme canbe the (L)-lactate dehydrogenase from Lactococcus lactis (Ll-LdhA), fromOryctalagus cuniculus (Oc-LldhA), from Geobacillus stearothermophilus(Gs-Lldh), or from Bacillus subtilis (Bs-Ldh), the (D)-lactatedehydrogenase from Escherichia coli (Ec-LdhA), the (L)-malatedehydrogenase from Escherichia coli (Ec-Mdh), or the branched chain(D)-2-hydroxyacid dehydrogenase from Lactococcus lactis (Ll-PanE).

In an even more specific aspect of the invention the OHB reductaseenzyme is represented by the amino acid sequences SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 288, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 102,SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ IDNO: 112, SEQ ID NO: 114, SEQ ID NO: 116 or SEQ ID NO: 118 or anysequence sharing a homology of at least 50% with said sequences, or isencoded by the nucleic acid sequences represented by SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 287, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 101,SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ IDNO: 111, SEQ ID NO: 113, SEQ ID NO: 115 or SEQ ID NO: 117 or anysequence sharing a homology of at least 50% with said sequences.

In a further aspect, the invention also deals with the use of an enzymereducing OHB to 2,4-DHB as above described.

Proteins sharing substantial homology with the above enzymes are alsoanother aspect of the invention such as functional variants orfunctional fragments.

The expression “substantial homology” covers homology with respect tostructure and/or amino acid components and/or biological activity.

More generally, within the meaning of the invention the homology betweentwo protein sequences can be determined by methods well known by theskilled man in the art. It is generally defined as a percentage ofsequence identity between a reference sequence and the sequence of aprotein of interest.

As used herein, “percent (%) sequence identity” with respect to theamino acid or nucleotide sequences identified herein is defined as thepercentage of amino acid residues or nucleotides in a candidate sequencethat are identical with the amino acid residues or nucleotides in anenzyme sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Methods for performing sequence alignment and determiningsequence identity are known to the skilled artisan, may be performedwithout undue experimentation, and calculations of identity values maybe obtained with definiteness. See, for example, Ausubel, et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 19 (GreenePublishing and Wiley-Interscience, New York); and the ALIGN program(Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3(National Biomedical Research Foundation, Washington, D.C.). A number ofalgorithms are available for aligning sequences and determining sequenceidentity and include, for example, the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443; the local homologyalgorithm of Smith, et al. (1981) Adv. Appl. Math. 2:482; the search forsimilarity method of Pearson, et al. (1988) Proc. Natl. Acad. Sci.85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187(1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul, et al.(1990) J. Mol. Biol. 215:403-410). Computerized programs using thesealgorithms are also available, and include, but are not limited to:ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul, et al.,Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST (Altschul, etal.), supra, FASTA, and TFASTA, available in the Genetics ComputingGroup (GCG) package, Version 8, Madison, Wis., USA; and CLUSTAL in thePC/Gene program by Intelligenetics, Mountain View, Calif. Those skilledin the art can determine appropriate parameters for measuring alignment,including algorithms needed to achieve maximal alignment over the lengthof the sequences being compared. Preferably, the sequence identity isdetermined using the default parameters determined by the program.Specifically, sequence identity can be determined by the Smith-Watermanhomology search algorithm (Meth. Mol. Biol. 70:173-187 (1997)) asimplemented in MSPRCH program (Oxford Molecular) using an affine gapsearch with the following search parameters: gap open penalty of 12, andgap extension penalty of 1. Preferably, paired amino acid comparisonscan be carried out using the GAP program of the GCG sequence analysissoftware package of Genetics Computer Group, Inc., Madison, Wis.,employing the blosum 62 amino acid substitution matrix, with a gapweight of 12 and a length weight of 2. With respect to optimal alignmentof two amino acid sequences, the contiguous segment of the variant aminoacid sequence may have additional amino acid residues or deleted aminoacid residues with respect to the reference amino acid sequence. Thecontiguous segment used for comparison to the reference amino acidsequence will include at least 20 contiguous amino acid residues, andmay be 30, 40, 50, or more amino acid residues. Corrections forincreased sequence identity associated with inclusion of gaps in thederivative's amino acid sequence can be made by assigning gap penalties.

The enzymes according to the present invention having the same activity(either OHB reductase, or the enzyme converting the primary amino groupof homoserine to a carbonyl group to obtain OHB) share at least about50%, 70% or 85% amino acid sequence identity, preferably at least about85% amino acid sequence identity, more preferably at least about 90%amino acid sequence identity, even more preferably at least about 95%amino acid sequence identity and yet more preferably 98% amino acidsequence identity. Preferably, any amino acid substitutions are“conservative amino acid substitutions” using L-amino acids, wherein oneamino acid is replaced by another biologically similar amino acid.Conservative amino acid substitutions are those that preserve thegeneral charge, hydrophobicity/hydrophilicity, and/or steric bulk of theamino acid being substituted. Examples of conservative substitutions arethose between the following groups: Gly/Ala, Val/Ile/Leu, Lys/Arg,Asn/Gln, Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr. A derivative may, forexample, differ by as few as 1 to 10 amino acid residues, such as 6-10,as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The term functional variant encompasses enzymes that may presentsubstantial sequence modifications when compared to the sequencesspecifically described within the present application but that stillretain the original enzymatic activity.

It also means that the sequence of the enzyme may comprise less aminoacids than the original one but said truncated enzyme still retains theoriginal enzymatic activity.

According to an aspect of the invention, the activity of the enzymecatalyzing the first and/or the second step of the method of the presentinvention is enhanced. This enhancement can be measured by an enzymaticassay as described in Examples 1 or 4.

Improvement of said enzymes can be obtained by at least one mutation,said mutation(s) (i) improving the activity and/or substrate affinity ofthe mutated enzyme for homoserine or OHB respectively, and or (ii)decreasing the activity and/or substrate affinity of the mutated enzymefor their natural substrate.

Within the present invention, the expression “improve the activityand/or substrate affinity” means that the enzyme before mutation, waseither

-   -   unable to use the substrate, and/or    -   synthesized the product of the reaction at a maximum specific        rate at least three times lower, and/or    -   had an affinity for homoserine or OHB that was at least three        times lower, and/or.    -   had a maximum specific activity on the natural substrate that        was at least three times higher, and/or.    -   had an affinity for the natural substrate that was at least        three times higher.

In a still further aspect the invention encompasses the nucleotidesequences encoding the enzymes catalyzing the first and the second stepof the method of the invention.

In an even more specific aspect of the invention the OHB reductaseenzyme is encoded by the nucleic acid sequences represented by SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 287, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115 or SEQ ID NO: 117 orany sequence sharing a homology of at least 50% with said sequences.

The OHB reductase according to the invention corresponds in a specificaspect to (L)-lactate dehydrogenase A comprising at least one mutationwhen a compared to the wild type enzyme in at least one of the positionsV17, Q85, E89, I226, or A222. These positions are conserved in thelactate dehydrogenase family, and they are defined in this text byreference to the Lactococcus lactis (L)-lactate dehydrogenase A (SEQ IDNO: 6). The skilled man in the art will then easily identify thecorresponding amino acid residues in other lactate dehydrogenases by analignment of the corresponding amino acid sequences. Therefore, theinvention also provides for changes of these amino acids in otherlactate dehydrogenase enzymes. 2

The OHB reductase according to the invention corresponds in a specificaspect to (L)-malate dehydrogenase comprising at least one mutation whencompared to the wild type enzyme in at least one of the positions I12,R81, M85, D86, V93, G179, T211, or M227. These positions are conservedin the malate dehydrogenase family, and they are defined in this text byreference to the sequence of the E. coli (L)-malate dehydrogenase (SEQID NO: 2). The man skilled in the art will easily identify thecorresponding amino acid residues in other malate dehydrogenases by analignment of the corresponding amino acid sequences. Therefore, theinvention also provides for changes of these amino acids in other malatedehydrogenase enzymes.

In accordance with this invention, a “nucleic acid sequence” refers to aDNA or RNA molecule in single or double stranded form, preferably a DNAmolecule. An “isolated DNA”, as used herein, refers to a DNA which isnot naturally-occurring or no longer in the natural environment whereinit was originally present, e.g., a DNA coding sequence associated withother regulatory elements in a chimeric gene, a DNA transferred intoanother host cell, or an artificial, synthetically-made DNA sequencehaving a different nucleotide sequence compared to anynaturally-occurring DNA sequence.

The present invention also relates to a chimeric gene comprising,functionally linked to one another, at least one promoter which isfunctional in a host organism, a polynucleotide encoding anyone of theenzymes catalyzing first and second step of the method as definedaccording to the invention, and a terminator element that is functionalin the same host organism. The various elements which a chimeric genemay contain are, firstly, elements regulating transcription, translationand maturation of proteins, such as a promoter, a sequence encoding asignal peptide or a transit peptide, or a terminator elementconstituting a polyadenylation signal and, secondly, a polynucleotideencoding a protein. The expression “functionally linked to one another”means that said elements of the chimeric gene are linked to one anotherin such a way that the function of one of these elements is affected bythat of another. By way of example, a promoter is functionally linked toa coding sequence when it is capable of affecting the expression of saidcoding sequence. The construction of the chimeric gene according to theinvention and the assembly of its various elements can be carried outusing techniques well known to those skilled in the art. The choice ofthe regulatory elements constituting the chimeric gene dependsessentially on the host organism in which they must function, and thoseskilled in the art are capable of selecting regulatory elements whichare functional in a given host organism. The term “functional” isintended to mean capable of functioning in a given host organism.

The promoters which the chimeric gene according to the invention maycontain are either constitutive or inducible. By way of example, thepromoters used for expression in bacteria may be chosen from thepromoters mentioned below. For expression in Escherichia coli mentionmay be made of the lac, trp, lpp, phoA, recA, araBAD, prou, cst-I, tetA,cadA, nar, tac, trc, lpp-lac, Psyn, cspA, PL, PL-9G-50, PR-PL, T7,[lambda]PL-PT7, T3-lac, T5-lac, T4 gene 32, nprM-lac, VHb and theprotein A promoters or else the Ptrp promoter (WO 99/64607). Forexpression in Gram-positive bacteria such as Corynebacteria orStreptomyces, mention may be made of the PtipA or PS1 and PS2(FR91/09870) promoters or those described in application EP0629699A2.For expression in yeasts and fungi, mention may be made of the K. lactisPLAC4 promoters or the K. lactis Ppgk promoter (patent application FR91/05294), the Trichoderma reesei tef1 or cbh1 promoter (WO 94/04673),the Penicillium funiculosum his, csl or apf promoter (WO 00/68401) andthe Aspergillus niger gla promoter.

According to the invention, the chimeric gene may also comprise otherregulatory sequences, which are located between the promoter and thecoding sequence, such as transcription activators (enhancers).

As such, the chimeric gene of the invention comprises in a specificembodiment at least, in the direction of transcription, functionallylinked, a promoter regulatory sequence which is functional in a hostorganism, a nucleic acid sequence encoding a polynucleotide encodinganyone of the enzymes catalyzing first and second step of the method asdefined according to the invention and a terminator regulatory sequencewhich is functional in said host organism.

The present invention also relates to a cloning and/or expression vectorcomprising a chimeric gene according to the invention or a nucleic acidsequence of the invention. The vector according to the invention is ofuse for transforming a host organism and expressing in this organismanyone of the enzymes catalyzing the first and/or the second step(s) ofthe method of the present invention. This vector may be a plasmid, acosmid, a bacteriophage or a virus. Preferentially, the transformationvector according to the invention is a plasmid. Generally, the mainqualities of this vector should be able to maintain itself and toself-replicate in the cells of the host organism, in particular byvirtue of the presence of an origin of replication, and to expressanyone of the enzymes catalyzing the first and/or the second step(s) ofthe method of the present invention therein. For the purpose of stabletransformation of a host organism, the vector may also integrate intothe genome. The choice of such a vector, and also the techniques ofinsertion of the chimeric gene according to the invention into thisvector are part of the general knowledge of those skilled in the art.Advantageously, the vector used in the present invention also contains,in addition to the chimeric gene according to the invention, a chimericgene encoding a selectable marker. This selectable marker makes itpossible to select the host organisms which are effectively transformed,i.e. those which incorporated the vector. According to a particularembodiment of the invention, the host organism to be transformed is abacterium, a yeast, a fungus. Among the selectable markers which can beused, mention may be made of markers containing genes for resistance toantibiotics, such as, for example, the hygromycinphosphotransferasegene. Other markers may be genes to complement an auxotrophy, such asthe pyrA, pyrB, pyrG, pyr4, arg4, argB and trpC genes, the molybdopterinsynthase gene or that of acetamidase. Mention may also be made of genesencoding readily identifiable enzymes such as the GUS enzyme, or genesencoding pigments or enzymes regulating the production of pigments inthe transformed cells. Such selectable marker genes are in particulardescribed in patent applications WO 91/02071, WO 95/06128, WO 96/38567and WO 97/04103.

The present invention also relates to modified microorganisms.

More specifically, the modified microorganism of the invention allowsthe preparation of 2,4-DHB from homoserine by a two-step pathwaycomprising:

-   -   a first step of conversion of the primary amino group of        homoserine to a carbonyl group to obtain        2-oxo-4-hydroxybutyrate, and    -   a second step of reduction of the obtained        2-oxo-4-hydroxybutyrate to obtain 2,4-dihydroxybutyrate.

The enzymes involved in the two steps are those above described.

The term “microorganism” is intended to mean any lower unicellularorganism into which the chimeric gene(s), nucleic acid(s) or vector(s)according to the invention may be introduced in order to produce2,4-DHB. Preferably, the host organism is a microorganism, in particulara fungus, for example of the Penicillium, Aspergillus and moreparticularly Aspergillusflavus, Chrysosporium or Trichoderma genus, ayeast, in particular of the Saccharomycetaceae, Pichiaceae orSchizosaccharomycetaceae, most preferentially Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromycesmarxianus, or Pichia jadinii, Pichia stipitis or Pichia pastoris, abacterium, preferentially selected among Enterobacteriaceae,Clostridiaceae, Bacillaceae, Streptomycetaceae, Streptococcaceae,Methylobacteriacae, and Corynebacteriaceae, most preferentiallyEscherichia coli, Bacillus subtilis, Corynebacterium glutamicum,Clostridium acetobutylicum, Methylobacterium extorquens or Lactococcuslactis.

The present invention also relates to modified microorganisms containingat least one chimeric gene according to the invention, either integratedinto their genome or carried on an extra-chromosomal genetic element,for example a plasmid. In a more specific aspect of the invention, thetransformed host organism comprises a nucleic acid of the inventionencoding a polypeptide converting the primary amino acid group ofhomoserine to a carbonyl group to obtain OHB and/or a nucleic acidencoding a polypeptide reducing OHB in 2,4-DHB or a chimeric genecomprising a nucleic acid encoding a polypeptide converting the primaryamino acid group of homoserine to a carbonyl group to obtain OHB, and/ora OHB reductase or an expression vector comprising a nucleic acidencoding a polypeptide converting the primary amino acid group ofhomoserine to a carbonyl group to obtain OHB, or a polypeptide having aOHB reductase activity.

Within a further aspect of the invention, the synthetic pathway for theconversion of homoserine into DHB is expressed in a microorganism withenhanced production of homoserine. Enhanced production of homoserine inmicroorganisms can be achieved by (i) overexpressing the enzymesaspartate kinase, aspartate semialdehyde dehydrogenase, and homoserinedehydrogenase, (ii) by rendering the aspartate kinase enzyme insensitiveto product inhibition that can be brought about by lysine, methionine,or threonine, and (iii) by deletion of metabolic pathways that branchoff the homoserine biosynthesis pathway. Overexpression of aspartatekinase, aspartate semialdehyde dehydrogenase, and homoserinedehydrogenase can be achieved by expressing the enzymes from a multicopyplasmid under the control of an appropriate constitutive or induciblepromoter. Alternatively, overexpression of said enzymes can be achievedby deletion of transcriptional repressors that limit the transcriptionof genes coding for aspartate kinase, aspartate semialdehydedehydrogenase, and homoserine dehydrogenase. Aspartate kinases can berendered insensitive to inhibition by aspartate-derived amino acids byintroducing appropriate mutations into their amino acid sequences. Entrypoints into metabolic pathways that branch off the homoserinebiosynthesis pathway are catalyzed by enzymes having O-succinylhomoserine or O-acetyl homoserine synthase activity (entry intomethionine biosynthesis), homoserine kinase activity (entry intothreonine biosynthesis), or diaminopimelate decarboxylase activity(entry into lysine biosynthesis). Deletion of genes encoding proteinshaving said enzymatic activities avoids formation aspartate-derivedamino acids and therefore aids homoserine formation.

Accordingly, deletion of the genes metA, thrB, and lysA in E. coliattenuates pathways that branch of the homoserine biosynthetic pathway.The increase of enzymatic activities of the homoserine pathway in E.coli can be achieved, for instance, by the overexpression of thebifunctional aspartate kinase-homoserine dehydrogenase mutant thrA S345F(insensitive to threonine inhibition) and asd (both genes from E. coli);or by the overexpression of the monofunctional aspartate kinase mutantlysC E250K (insensitive to lysine), asd (both genes from E. coli), andthe homoserine dehydrogenase gene HOM6 from S cerevisiae.

The microorganism of the invention may also have attenuated capacity toexport homoserine which increases the intracellular availability of thisamino acid. In order to achieve decreased homoserine export from thecells, permeases capable of exporting homoserine can be deleted. Suchpermeases may be identified by overexpressing genomic libraries in themicroorganism and cultivating said microorganism at inhibitoryconcentrations of homoserine or structurally similar amino acids such asthreonine, leucine, or aspartate (Zakataeva et al. 1999/FEBSLett/452/228-232). Genes whose overexpression confers growth atincreased concentrations of either of said amino acids are likely toparticipate in homoserine export.

In a further aspect, the microorganism of the invention beingEscherichia coli carries deletions in the homoserine efflux transportersrhtA, rhtb, and/or rhtC.

Efficient production of DHB can be ensured by optimizing carbon fluxrepartitioning in the metabolic network of the host organism withrespect to the optimization of cofactor supply for DHB synthesis, andattenuation of competing pathways that cause formation of metabolicby-products other than DHB. An important tool for strain improvementprovides constraint-based flux balance analysis. This method allowscalculating the theoretical yield of a given metabolic network dependingon cultivation conditions, and facilitates identification of metabolictargets for overexpression or deletion. The experimental techniques usedfor overexpression and deletion of the metabolic target reaction aredescribed (Example 8).

Accordingly, the microorganism of the invention may also exhibitenzymatic activities chosen among phosphoenolpyruvate carboxylase,phosphoenolpyruvate carboxykinase, isocitrate lyase, pyruvatecarboxylase, and hexose symporter permease which is increased, and/or atleast one of the enzymatic activities chosen among lactatedehydrogenase, alcohol dehydrogenase, acetate kinase, phosphateacetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase,2-oxoglutarate dehydrogenase, pyruvate kinase, malic enzyme,phosphoglucose isomerase, phosphoenolpyruvate carboxylase,phosphoenolpyruvate carboxykinase, pyruvate-formate lyase, succinicsemialdehyde dehydrogenase, sugar-transporting phosphotransferase,ketohydroxyglutarate aldolase, homoserine-O-succinyl transferase,homoserine kinase, homoserine efflux transporter, diaminopimelatedecarboxylase, and/or methylglyoxal synthase which is (are) decreased.

In a further aspect, the microorganism of the invention beingEscherichia coli overexpresses at least one of the genes chosen amongppc, pck, aceA, galP, asd, thrA, metL, lysC all E. coli; pycA from L.lactis, and/or has at least one of the genes deleted chosen among ldhA,adhE, ackA, pta, poxB, focA, pfIB, sad, gabABC, sfcA, maeB, ppc, pykA,pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABC, aldA, lldD, icIR, metA,thrB, lysA, eda, rhtA, rhtB, rhtC.

The present invention also encompasses a method of production of 2,4-DHBcomprising the steps of

-   -   culturing the modified microorganism of the invention in an        appropriate culture medium,    -   recovering 2,4-DHB from the culture medium. Said 2,4-DHB can be        further purified.

Product separation and purification is very important factor enormouslyaffecting overall process efficiency and product costs. Methods forproduct recovery commonly comprise the steps cell separation, as well asproduct purification, concentration and drying, respectively.

Cell Separation

Ultrafiltration and centrifugation can be used to separate cells fromthe fermentation medium. Cell separation from fermentation media isoften complicated by high medium viscosity. Therefore, we can addadditives such as mineral acids or alkali salts, or heating of theculture broth to optimize cell separation.

Product Recovery

A variety of ion-exchange chromatographic methods can be applied for theseparation of DHB either before or after biomass removal. They includethe use of primary cation exchange resins that facilitate separation ofproducts according to their isoelectric point. Typically, the resin ischarged with the solution, and retained product is eluted separatelyfollowing increase of pH (e.g. by adding ammonium hydroxide) in theeluent. Another possibility is the use of ion-exchange chromatographyusing fixed or simulated moving bed resins. Different chromatographicsteps may have to be combined in order to attain adequate productpurity. Those purification methods are more economical compared with acostly crystallization step, also providing additional advantages andflexibility regarding the form of final product.

Product Concentration and Drying

The purification process can also comprise a drying step which mayinvolve any suitable drying means such as a spray granulator, spraydryer, drum dryer, rotary dryer, and tunnel dryer. Concentrated DHBsolutions can be obtained by heating fermentation broths under reducedpressure by steam at 130° C. using a multipurpose concentrator or thinfilm evaporator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Method of preparation of 2,4-DHB from homoserine comprising atwo step pathway which employs a first step of conversion of the primaryamino group of homoserine to a carbonyl group to obtain OHB, and asecond step of reduction of the obtained OHB to 2,4-DHB.

FIG. 2: Specific activities of purified L. lactis lactate dehydrogenasemutated in position Q85. (A) specific activities on OHB, (B) specificactivities on pyruvate, (C) Substrate specificity expressed as ratio ofVmax values on OHB and pyruvate. Values higher than 1 in graph Cindicate preference for OHB (no saturation of enzymatic activity wasobtained on either substrate for mutated enzymes between 0 and 50 mM OHBor pyruvate). Activities were measured at a substrate concentration of20 mM.

FIG. 3: Specific activities of purified E. coli malate dehydrogenasemutated in position R81. (A) specific activities on OHB, (B) specificactivities on oxaloacetate. Activities were measured at a substrateconcentration of 20 mM OHB or 0.5 mM oxaloacetate.

The following non limiting examples illustrate the invention.

EXAMPLES Example 1 Demonstration of OHB Reductase Activity

Construction of plasmids containing wild-type genes coding for lactatedehydrogenase or malate dehydrogenase:

The genes coding for (L)-malate dehydrogenase in Escherichia coli,Ec-mdh (SEQ ID NO: 1), (D)-lactate dehydrogenase in E. coli, Ec-ldhA(SEQ ID NO: 3), (L)-lactate dehydrogenase of Lactococcus lactis, Ll-ldhA(SEQ ID NO: 5), (L)-lactate dehydrogenase of Bacillus subtilis, Bs-ldh(SEQ ID NO: 7), (L)-lactate dehydrogenase of Geobacillusstearothermophilus, Gs-ldh (SEQ ID NO: 9), the two isoforms of the(L)-lactate dehydrogenase of Oryctalagus cuniculus, Oc-ldhA (SEQ ID NO:11 and SEQ ID NO: 13), were amplified by PCR using the high-fidelitypolymerase Phusion™ (Fermentas) and the primers listed in Table 1.Genomic DNAs of E. coli MG1655, L. lactis IL1403, and B. subtilis strain168 were used as the template. The genes Oc-ldhA, and Gs-ldh werecodon-optimized for expression in E. coli and synthesized by MWGEurofins. The primers introduced restriction sites (Table 1) upstream ofthe start codon and downstream of the stop codon, respectively,facilitating the ligation of the digested PCR products into thecorresponding sites of the pET28a+ (Novagen) expression vector using T4DNA ligase (Fermentas). Ligation products were transformed into E. coliDH5a cells (NEB). The resulting pET28-Ec-mdh, pET28-Ec-ldhA,pET28-Ll-ldhA, pET28-Bs-ldh, pET28-Gs-ldh, and pET28-Oc-ldhA plasmidswere isolated and shown by DNA sequencing to contain the correctfull-length sequence of the E. coli mdh, E. coli ldhA, L. lactis ldhA,B. subtilis ldh, G. stearothermophilus ldh, and O. cuniculus ldhA genes,respectively. The corresponding protein sequences are represented by SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12 and SEQ ID NO: 14, respectively.

TABLE 1 Primer sequences and restrictionsites used for amplification and cloning of candidate enzymesForward and reverse Restriction Gene primer sequence 5′ - 3′ sitesEc-mdh TATAATCATATGAAAGTCGCAGTCCTC NdeI (SEQ ID No 15). BamHITATAATGGATCCTTACTTATTAACGAACT C (SEQ ID No. 16) Ll-ldhATATAATCATATGGCTGATAAACAACGTAA NdeI AAAA (SEQ ID No. 17) BamHITATAATGGATCCTTAGTTTTTAACTGCAG AAGCAAA (SEQ ID No. 18) Bs_ldhTATAATGCTAGCATGATGAACAAACATGT NdeI AAATAAAGT (SEQ ID No. 19) BamHITATAATGGATCCTTAGTTGACTTTTTGTT C (SEQ ID No. 20) Gs-ldhGene was delivered by MWG NdeI Eurofins™ in pET28a vector BamHI Oc-ldhATATAATGCTAGCATGGCGGCGTTGAAAGA NheI C (SEQ ID No. 21) EcoRIATTATAGAATTCTTAAAATTGCAGTTCTT T (SEQ ID No. 22) Ll-panETATAATCATATGAGAATTACAATTGCCGG NdeI (SEQ ID No. 23) BamHITATAATGGATCCTTATTTTGCTTTTAATA ACTCTTCTTTGC (SEQ ID No. 24) Ec-ldhATATAATCATATGAAACTCGCCGTTTATAG NdeI (SEQ ID No. 25) BamHITATAATGGATCCTTAAACCAGTTCGTTCG G (SEQ ID No. 26)

Expression of enzymes: E. coli BL21 (DE3) star cells were transformedwith the appropriate plasmids using standard genetic protocols(Sambrook, Fritsch, & Maniatis, 1989). Enzymes with an N-terminalhexa-His tag were expressed in 50 mL LB cultures that were inoculatedfrom an overnight culture at OD₆₀₀ of 0.1 and grown to OD₆₀₀ of 0.6before protein expression was induced by addition of 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) to the culture medium. After 15 h ofprotein expression, cells were harvested by centrifugation at 4000 g at4° C. for 10 min and discarding the supernatant. Cell pellets werestored at −20° C. until further analysis. Growth and protein expressionwere carried out at 25° C. Culture media contained 50 μg/mL kanamycin.

Purification of enzymes: Frozen cell pellets of expression cultures wereresuspended in 0.5 mL of breakage buffer (50 mM Hepes, 300 mM NaCl, pH7.5) and broken open by four successive rounds of sonication (sonicationinterval: 20 s, power output: 30%, sonicator: Bioblock Scientific,VibraCell™ 72437). Cell debris was removed by centrifuging the crudeextracts for 15 min at 4° C. at 4000 g and retaining the clearsupernatant. RNA and DNA were removed from the extracts by adding 15mg/mL streptomycin sulfate (Sigma), centrifuging the samples at 13000 gfor 10 min at 4° C. and retaining the supernatant. Clear protein extractwas incubated for 1 h at 4° C. with 0.75 mL (bed volume) of Talon™Cobalt affinity resin (Clontech). The suspension was centrifuged at 700g in a table top centrifuge and supernatant was removed. The resin waswashed with 10 bed volumes of wash buffer (50 mM Hepes, 300 mM NaCl, 15mM Imidazole, pH 7.5) before proteins were eluted with 0.5 mL of elutionbuffer (50 mM Hepes, 300 mM NaCl, 250 mM Imidazole, pH 7.5). Purity ofeluted enzymes was verified by SDS-PAGE analysis. Protein concentrationswere estimated with the method of Bradford (Bradford (1976, Anal.Biochem. 72: 248-54). To stabilize the lactate dehydrogenase enzymes,the elution buffer was systematically exchanged by 100 mM phosphatebuffer adjusted to pH 7. The protein sample was transferred to anAmicon™ Ultra centrifugal filter (cut-off 10 kDa), and centrifugedduring 8 min at 4000 g at 4° C. to remove the buffer. The protein wasdiluted into phosphate buffer and the procedure was repeated 4 times.

Enzymatic assays: The reaction mixture contained 60 mM Hepes (pH 7), 50mM potassium chloride, 5 mM MgCl₂, 0.25 mM NADH, (optionally 5 mMfructose-1,6-bisphosphate) (all products from Sigma), and appropriateamounts of purified malate or lactate dehydrogenase or cell extract.Reactions were started by adding appropriate amounts of2-oxo-4-hydroxybutyrate (OHB), pyruvate, or oxaloacetate (OAA).Enzymatic assays were carried out at 37° C. in 96-well flat bottomedmicrotiter plates in a final volume of 250 μL. The reactions werefollowed by the characteristic absorption of NADH at 340 nm(ε_(NADH)=6.22 mM⁻¹ cm⁻¹) in a microplate reader (BioRad 680XR).

OHB was synthesized by incubating 125 mM homoserine with snake venom(L)-amino acid oxidase (1.25 U/mL, Sigma) and catalase (4400 U/mL,Sigma) in 100 mM Tris buffer at pH 7.8 for 90 min at 37° C.Subsequently, the reaction mixture was purified on an Amicon™ Ultracentrifugal filter with a cut-off of 10 kDa to eliminate the enzymes(method adapted from Wellner & Lichtenberg, 1971).

OHB was quantified by mixing 100 μL of the tested solution with 1 mL ofa solution containing 1 M sodium arsenate and 1 M boric acid at pH 6.5.The mixture was incubated at room temperature for 30 min and theabsorbance at 325 nm was used to quantify OHB. The relation betweenabsorbance and concentration of the ketone was calibrated using pyruvatesolutions of known concentrations (method adapted from (Wellner &Lichtenberg, 1971)). The typical OHB yield of the method was 90%.

Results: The kinetic parameters are listed in Table 2 for the testedenzymes on their natural substrates and OHB. Significant OHB reductaseactivity was found for all lactate dehydrogenases of differentbiological origin. Malate dehydrogenase, Mdh, of E. coli only had veryminor activity on OHB. The branched chain 2-oxo-acid dehydrogenase,PanE, from L. lactis also had significant activity on OHB.

TABLE 2 Summary of kinetic parameters of selected candidate enzymes ontheir natural substrate and OHB Max. specific activity Substrateaffinity, Km [μmol/(mg min)] [mM] Natural Natural Enzyme substrate^(a)OHB^(b) substrate^(a) OHB Ec-Mdh 95.6 0.01  0.04 ns Ll-Ldh 184 18 2.7 nsGs-Ldh 87.7 66.8 1.2 1.3 Bs-Ldh 170 15.7 nd ns Ll-PanE nd 2.58 nd nsOc-LdhA 68.3 6.5 1.5 13   Ec-LdhA 265 0.56 1.8 4.8 ^(a)Naturalsubstrates for Mdh and Ldh are oxaloacetate and pyruvate, respectively^(b)When enzymes could not be saturated, maximum specific activityrefers to the activity estimated at 20 mM substrate concentration ns—notsaturated nd—not determined

Example 2 Construction of Lactate Dehydrogenase Enzymes with ImprovedOHB Reductase Activity

Site-directed mutagenesis of the L. lactis ldhA gene was carried outusing the pET28-Ll-ldhA plasmid as the template. Point mutations tochange the amino acid sequence were introduced by PCR (Phusion 1U, HFbuffer 20% (v/v), dNTPs 0.2 mM, direct and reverse primers 0.04 μM each,template plasmid 30-50 ng, water) using the oligonucleotide pairs listedin Table 3. The genes mutated by PCR contained a new restriction sitelisted in Table 3 (introduced using silent mutations) in addition to thefunctional mutation to facilitate identification of mutated clones. ThePCR products were digested by DpnI at 37° C. for 1 h to remove templateDNA, and transformed into competent E. coli DH5α (NEB) cells. Themutated plasmids were identified by restriction site analysis and wereverified to carry the desired mutations by DNA sequencing.

TABLE 3 Oligonucleotides used to mutate lactate dehydrogenase ldhA fromL. lactis (nnk denotes a degenerated codonwith k representing either thymine or cytosine) Primer sequencesRestriction Protein Mutation 5′ - 3′ site Ll-LdhA Q85nnkGTCTTGACTTCTGGTG MluI CTCCANNKAAACCAGG TGAAACGCGTCTT (SEQ ID NO. 27)AAGACGCGTTTCACCT GGTTTMNNTGGAGCAC CAGAAGTCAAGAC  (SEQ ID NO. 28) Ll-LdhAI226V CGTGATGCTGCTTACT PvuI CGATCGTCGCTAAAAA AGGTG  (SEQ ID No. 99)CACCTTTTTTAGCGAC GATCGAGTAAGCAGCA TCACG  (SEQ ID No. 100)Mutant enzymes were expressed, purified and tested for OHB and pyruvatereductase activity as described in Example 1. The activity measurementsfor both substrates are summarized in FIG. 2. The results demonstratethat the replacement of Gln85 by preferably alanine, cysteine,asparagine, or methionine yields an increase of the enzyme's specificityfor OHB, and/or an increase in maximum specific OHB reductase activity.The mutation Q85N in Ll-Ldh was combined with mutation I226V. It wasdemonstrated that this exchange had a major positive impact on substrateaffinity for OHB.

TABLE 4 Summary of kinetic parameters of L. lactis lactate dehydrogenaseA, Ll-LdhA, mutants on pyruvate and OHB Max. specific activity Km Mutant[μmol/(mg min)] [mM] Enzyme Seq ID Pyruvate OHB Pyruvate OHB Q85N SEQ IDNo. 30 184 63.9 22.1 29.2 Q85NI226V SEQ ID No. 32 11.5 4.9 1.4 3.3

Example 3 Construction of Malate Dehydrogenase Enzymes with Improved OHBReductase Activity

Site-directed mutagenesis of the mdh gene from E. coli was carried outas described in Example 2 using the primers listed in Table 5. PlasmidpET28-Ec-mdh was used as the template.

TABLE 5 Oligonucleotides used to mutatemalate dehydrogenase mdh from E. coli.(nnk denotes a degenerated codon with krepresenting either thymine or cytosine) Primer sequences Restr. ProteinMutation 5′ - 3′ site Ec-Mdh R81nnk TTATCTCTGCAGGCGT Sma1AGCGNNKAAACCCGGG ATGGATCGTTC (SEQ ID No. 33) GAACGATCCATCCCGGGTTTMNNCGCTACGCC TGCAGAGATAA (SEQ ID No. 34) Ec-Mdh R81AM85ETTATCTCTGCAGGCGT no AGCGGCTAAACCGGGT Sma1 GAGGATCGTTCCGACC TG(SEQ ID NO. 35) CAGGTCGGAACGATCC TCACCCGGTTTAGCCG CTACGCCTGCAGAGAT AA(SEQ ID NO. 36) Ec-Mdh R81AM85Q TTATCTCTGCAGGCGT no AGCGGCTAAACCGGGTSma1 CAGGATCGTTCCGACC TG (SEQ ID NO. 37) CAGGTCGGAACGATCCTGACCCGGTTTAGCCG CTACGCCTGCAGAGAT AA  (SEQ ID NO. 38) Ec-Mdh I12VGTCGCAGTCCTCGGCG Nar1 CCGCTGGCGGTGTCGG CCAGGCGCTTGCAC  (SEQ ID NO. 39GTGCAAGCGCCTGGCC GACACCGCCAGCGGCG CCGAGGACTGCGAC  (SEQ ID NO. 40) Ec-MdhG179D CCG GTT ATT GGC Eae1 GGC CAC TCT GAT GTT ACC ATT CTG CCG CTG CTG(SEQ ID NO. 41) CAGCAGCGGCAGAATG GTAACATCAGAGTGGC CGCCAATAACCGG (SEQ ID NO. 42) Ec-Mdh R81AD86S GGCGTAGCGGCTAAAC no CGGGTATGTCTCGTTCSma1 CGACCTG (SEQ ID NO. 43) CAGGTCGGAACGAGAC ATACCCGGTTTAGCCG CTACGCC(SEQ ID NO. 44)Mutant enzymes were expressed, purified and tested for OHB andoxaloacetate reductase activity as described in Example 1. The activitymeasurements on OHB and oxaloacetate are summarized in FIG. 3. Theresults demonstrate that replacement of Arg81 by alanine, cysteine,glycine, histidine, isoleucine, leucine, methionine, asparagine,glutamine, serine, threonine, or valine confer significant OHB reductaseactivity, and concomitant decrease of oxaloacetate reductase activity.The mutation R81A in Ec-Mdh was combined with additional changes in theprotein sequence. The results are listed in Table 6. It was demonstratedthat the introduction of mutations M85Q, M85E, I12V, D86S or G179Dresult in an increased activity on OHB.

TABLE 6 Summary of kinetic parameters of E. coli malate dehydrogenasemutants on oxaloacetate (OAA) and OHB Max. specific activity Km Mutant[μmol/(mg min)] [mM] Enzyme Seq ID OAA^(a) OHB^(b) OAA OHB Wild-type SEQID No. 95 0.01 0.04 ns 2 R81A SEQ ID No. 1.16 1.8 ns ns 102 R81A SEQ IDNo. 0.5 4.99 ns ns M85Q 104 R81A SEQ ID No. 1 3 ns ns M85E 106 R81A SEQID No. 1.84 18.9 ns 15 M85Q I12V 108 R81A SEQ ID No. 2.2 12.54 ns nsM85E I12V 110 R81A SEQ ID No. 0.37 4.16 ns ns G179D 112 R81A D86S SEQ IDNo. 0.67 14.6 ns ns 1114 R81A 112V SEQ ID No. 0.5 4.9 ns ns 115 R81A SEQID No. 0.54 19 ns ns G179D 118 D86S ^(a)activity was measured at 0.5 mMoxaloacetate ^(b)activity was measured at 20 mM OHB ns—not saturated atconcentrations of up to 50 mM of OHB and 0.5 mM of oxaloacetate

Example 4 Demonstration of Homoserine Transaminase Activity for SelectedTransaminases

The genes coding for different transaminases in E. coli, S. cerevisiae,and L. lactis were amplified by PCR using the high-fidelity polymerasePhusion™ (Finnzymes) and the primers listed in Table 7. Genomic DNA ofE. coli MG1655, S. cerevisiae BY4741, and L. lactis IL1403 were used asthe templates. The primers introduced restriction sites (Table 7)upstream of the start codon and downstream of the stop codon,respectively, facilitating the ligation of the digested PCR productsinto the corresponding sites of the pET28a+(Novagen) expression vectorusing T4 DNA ligase (Biolabs). Ligation products were transformed intoE. coli DH5a cells. The resulting plasmids were isolated and shown byDNA sequencing to contain the correct full-length sequence of thecorresponding genes. The references to the corresponding proteinsequences are listed in Table 7.

TABLE 7 Primer sequences and restriction sitesused for amplification and cloning ofcandidate enzymes (Abbreviations used for sourceorganism: Ec - E. coli, Sc - S. cerevisiae,Ll - L. lactis). All the genes were cloned intopET28a+ (Novagen), adding an N-terminal Hexa-HisTag.Forward and reverse  Gene Protein Restriction Geneprimer sequences 5′ - 3′ sequence sequence sites Ec-ilvEtataatgctagcatgaccacgaagaaagctgattaca SEQ ID SEQ ID NheI (SEQ ID No. 47)No. 59 No. 60 BamHI tataatggatccttattgattaacttgatctaacc (SEQ ID No. 48)Ec-tyrB Tataatgctagcgtgtttcaaaaagttgacg SEQ ID SEQ ID NheI(SEQ ID No. 49) No. 61 No. 62 BamHI Tataatggatccttacatcaccgcagcaaac(SEQ ID No. 50) Ec-aspC Tataatgctagcatgtttgagaacattaccgc SEQ ID SEQ IDNheI (SEQ ID No. 51) No. 63 No. 64 BamHITataatggatccttacagcactgccacaatcg (SEQ ID No. 52) Ll-araTTataatgctagcatggatttattaaaaaaatttaaccctaa SEQ ID SEQ ID NheI(SEQ ID No. 53) No. 65 No. 66 BamHITataatggatcctcagccacgttttttagtcacataa (SEQ ID No. 54) Ll-bcaTTataatgctagcatggcaattaatttagactg SEQ ID SEQ ID NheI (SEQ ID No. 55)No. 67 No. 68 BamHI Tataatggatccttaatcaactttaactatcc (SEQ ID No. 56)Sc-ARO8 Tataatcatatgatcatgactttacctgaatcaaaaga SEQ ID SEQ ID NheI(SEQ ID No. 57) No. 69 No. 70 BamHITataatggatccctatttggaaataccaaattcttcg (SEQ ID No. 58)Enzymes were expressed and purified as described in Example 1, andtested for homoserine transaminase activity under the conditionsdescribed below.

Enzymatic assays: Transaminase activity of several candidateaminotransferases was quantified with 2-oxoglutarate as the amino groupacceptor. Transaminase reactions were carried out using homoserine andthe preferred amino acid of the enzymes. The reactions were followed bythe amino acid-dependent oxidation of NADH in the coupled dehydrogenasereaction.

Transaminase Assays (Reaction Scheme)

Transaminase: Amino acid+2-oxoglutarate→2-oxo-acid+glutamate

Dehydrogenase: 2-oxo-acid+NADH→2-hydroxy-acid+NAD⁺

The reaction mixture contained 60 mM Hepes (pH 7), 50 mM potassiumchloride, 5 mM MgCl₂, 4 mM 2-oxoglutarate, 0.1 mM pyridoxal-5′-phosphate(PLP), 0.25 mM NADH, (optionally 5 mM fructose-1,6-bisphosphate) (allproducts from Sigma), 4 Units/mL of auxiliary 2-hydroxyaciddehydrogenase, and appropriate amounts of purified aminotransferase orcell extract. The auxiliary dehydrogenase enzyme was purified PanE fromL. lactis in case of the amino acids phenylalanine and leucine(Chambellon, Rijnen, Lorquet, Gitton, van HylckamaVlieg, Wouters, &Yvon, 2009), malate dehydrogenase (Sigma) in case of aspartate, andrabbit muscle (L)-lactate dehydrogenase (Sigma) when homoserine was usedas the starting substrate. Reactions were started by adding 50 mM of theamino acid.

Enzymatic assays were carried out at 37° C. in 96-well flat bottomedmicrotiter plates in a final volume of 250 μL. The reactions werefollowed by the characteristic absorption of NAD(P)H at 340 nm(ε_(NADPH)=6.22 mM⁻¹ cm⁻¹) in a microplate reader (BioRad 680XR).

Results: The kinetic parameters of different aminotransferases arelisted in Table 8. Significant homoserine transaminase activity wasfound for the listed transaminase enzymes.

TABLE 8 Transaminase activities of tested candidate enzymes onhomoserine and their preferred amino acid substrate (Abbreviations usedfor source organism: Ec—E. coli, Sc—S. cerevisiae, Ll—L. lactis). Max.specific activity on different substrates [μmol/(min mg_(prot))] EnzymeHomoserine* Preferred amino acid Ec-IlvE 0.077 10.3^((L)) Ec-TyrB 0.0579.03^((P)) Ec-AspC 0.082 74.031^((A)) Ll-AraT 0.109 11.72^((P)) Ll-BcaT0.028 30.39^((L)) Sc-ARO8 0.076 20.5^((P)) *activity measured at 50 mMhomoserine,

Example 5 Construction of Plasmids for Overexpression of the HomoserinePathway Enzymes

Construction of the Plasmids pTAC-op-HMS1 and pACT3-op-HMS1

The plasmid pET28-LYSCwt was constructed by amplifying the lysC gene byPCR using high fidelity polymerase Phusion™ (Finnzymes) and the directand reverse primers 5′ CACGAGGTACATATGTCTGAAATTGTTGTCTCC3′ (SEQ ID NO:71) and 5′ CTTCCAGGGGATCCAGTATTTACTCAAAC3′ (SEQ ID NO: 72) thatintroduced a NdeI and BamHI restriction sites upstream of the startcodon and downstream of the stop codon, respectively. Genomic DNA fromE. coli MG1655 was used as the template. The PCR product was digestedwith NdeI and BamHI, ligated into the corresponding sites of the pET28a(Novagen) expression vector using T4 DNA ligase (Biolabs), andtransformed into E. coli DH5a cells. The resulting pET28-LYSCwt plasmidwas isolated and shown by DNA sequencing to contain the full-length lysCgene having the correct sequence (SEQ ID NO: 73).

Site-directed mutagenesis of lysC to alleviate inhibition by lysine wascarried out using the pET28-LYSCwt plasmid as the template. A pointmutation to change the amino acid sequence in position 250 fromglutamate to lysine (E250K, SEQ ID NO: 36) was introduced by PCR(Phusion 1U, HF buffer 20% (v/v), dNTPs 0.2 mM, direct and reverseprimers 0.04 μM each, template plasmid 50 ng, water) using theoligonucleotides 5′ GCGTTTGCCGAAGCGGCAAAGATGGCCACTTTTG3′ (SEQ ID NO: 74)and 5′ CAAAAGTGGCCATCTTTGCCGCTTCGGCAAACGC3′ (SEQ ID NO: 75). The PCRproduct (SEQ ID NO: 35) was digested by DpnI at 37° C. for 1 h to removetemplate DNA, and transformed into competent E. coli DH5 α (NEB) cells.The mutated plasmid pET28-LYSC* was identified by restriction siteanalysis and verified to carry the desired mutations by DNA sequencing.

The plasmid pET28-ASDwt was constructed by amplifying the asd gene of E.coli by PCR using high fidelity polymerase Phusion™ (Finnzymes) and thedirect and reverse primers 5′ TATAATGCTAGCATGAAAAATGTTGGTTTTATCGG3′ (SEQID NO: 76) and 5′ TATAATGGA-TCCTTACGCCAGTTGACGAAGC3′ (SEQ ID NO: 77)that introduced a NheI and BamHI restriction site upstream of the startcodon and downstream of the stop codon, respectively. Genomic DNA fromE. coli DH5 α was used as the template. The PCR product was digestedwith NheI and BamHI, ligated into the corresponding sites of the pET28a(Novagen) expression vector using T4 DNA ligase (Biolabs), andtransformed into E. coli DH5a cells. The resulting pET28-ASDwt plasmidwas isolated and shown by DNA sequencing to contain the full-length asdgene having the correct sequence (SEQ ID NO: 98).

The plasmid pET28-HOM6 wt was constructed by amplifying the HOM6 gene ofS. cerevisiae by PCR using high fidelity polymerase Phusion™ (Finnzymes)and the direct and reverse primers 5′ TATAATCATATGAGCACTAAAGTTGTTAATG3′(SEQ ID NO: 78) and 5′ TATAATGGATC-CCTAAAGTCTTTGAGCAATC3′ (SEQ ID NO:79) that introduced a NdeI and BamHI restriction site upstream of thestart codon and downstream of the stop codon, respectively. Genomic DNAfrom S. cerevisiae BY4741 was used as the template. The PCR product wasdigested with NdeI and BamHI, ligated into the corresponding sites ofthe pET28a (Novagen) expression vector using T4 ligase (Biolabs), andtransformed into E. coli DH5a cells. The resulting pET28-HOM6 wt plasmidwas isolated and shown by DNA sequencing to contain the full-length HOM6gene having the correct sequence (SEQ ID NO: 97).

The plasmid pET28-LYSC* was used as the backbone for the construction ofthe pTAC-op-HMS plasmid that enabled the expression oflysine-insensitive aspartate kinase, aspartate semialdehydedehydrogenase, and homoserine dehydrogenase from an inducible tacpromoter.

The asd gene was obtained by PCR from pET28-asdwt. The whole codingregion and part of the upstream region comprising the pET28 ribosomebinding site (rbs) and the in-frame N-terminal His-Tag were amplified byPCR using high fidelity polymerase Phusion™ (Finnzymes) and the directand reverse primers 5′ TATAAGGATCCGTTTAACTTTAAGAAGGAGATATACCATGGG3′ (SEQID NO: 80) and 5TATAAGAATTCTTACGCCAGTTGACGAAG3′ (SEQ ID NO: 81) thatintroduced a BamHI and EcoRI restriction site upstream of the rbs anddownstream of the stop codon, respectively. The PCR product was digestedwith BamHI and EcoRI, ligated into the corresponding sites ofpET28-LYSC*, using T4 DNA ligase (Biolabs), and transformed into E. coliDH5a cells. The resulting pET28-LYSC*-ASD plasmid was isolated and shownby DNA sequencing to have the correct sequence.

The HOM6 gene was obtained by PCR from pET28-HOM6 wt. The whole codingregion and part of the upstream region comprising the pET28 ribosomebinding site and the in-frame N-terminal His-Tag were amplified by PCRusing high fidelity polymerase Phusion™ (Finnzymes), the direct primer5′ TATAAGCGGCCGCGTTTAACTTTAAGAAGGAGATAT3′ (SEQ ID NO: 82), and thereverse primer 5′ TATAAACTCGAGCCTAAAGTCTTTGAGCAAT3′ (SEQ ID NO: 83) thatintroduced a NotI and a PspXI restriction site upstream of the rbs anddownstream of the stop codon, respectively. The PCR product was digestedwith NotI and PspXI, ligated into the corresponding sites ofpET28-LYSC*-ASD, using T4 DNA ligase (Biolabs), and transformed into E.coli DH5a cells. The resulting pET28-op-HMS1 plasmid was isolated andshown by DNA sequencing to have the correct sequence.

The 5′ upstream promoter region simultaneously regulating the expressionof the three genes (i.e. the T7 promoter in pET28a+) can be replacedwith any other promoter, inducible or constitutive, by digesting theplasmids with SphI and XbaI and cloning another promoter region withsuitable restriction sites.

In the present non-exclusive example, the T7 promoter of the pET28a+backbone was replaced by the artificial IPTG-inducible tac promoter (deBoer et al., 1983). The tac promoter was obtained from plasmid pEXT20(Dykxhoorn et al., 1996) by digesting this plasmid with SphI and XbaI.The DNA fragment containing the promoter was purified and cloned intoSphI and XbaI digested pET28-op-HMS1 obtaining pTAC-op-HMS1. Theresulting pTAC-op-HMS plasmid was isolated and shown by DNA sequencingto have the correct sequence.

The operon containing the coding sequences of lysC*, asd, and HOM6 wasPCR amplified from the plasmid pTAC-op-HMS1 using the primers5′-TATAAAGATCTTAGAAATAATTTTGTTTA-3′ (SEQ ID NO: 84) and5′-TATAATCTAGACTAAAGTCTTTGAGCAAT-3′ (SEQ ID NO: 85) which introduced aBgIII and a XbaI restriction site at the 5′ and the 3′ end,respectively, of the PCR fragment. The fragment was purified, digestedwith BgIII and XbaI and cloned into the corresponding sites of pACT3(Dykxhoorn et al., 1996) to obtain the vector pACT3-op-HMS1. Theresulting pACT3-op-HMS1 plasmid was isolated and shown by DNA sequencingto have the correct sequence.

Construction of the Plasmids pEXT20-op-HMS2 and pACT3-op-HMS2

The plasmid pET28-thrAwt was constructed by amplifying the E. coli thrAgene encoding bifunctional enzyme aspartate kinase/homoserinedehydrogenase I by PCR using high fidelity polymerase Phusion™(Finnzymes) and the direct and reverse primers5′-TATAATCATATGCGAGTGTTGAAGTTCG-3′ (SEQ ID NO: 86) and5′-TATAATGGATCCTCAGACTCCTAACTTCCA-3′ (SEQ ID NO: 87) that introduced aNdeI and BamHI restriction sites upstream of the start codon anddownstream of the stop codon, respectively. Genomic DNA from E. coliMG1655 was used as the template. The PCR product was digested with NdeIand BamHI, ligated into the corresponding sites of the pET28a+(Novagen)expression vector using T4 DNA ligase (Biolabs), and transformed intoNEB 5-alpha competent E. coli cells (NEB). The resulting pET28-thrAwtplasmid was isolated and shown by DNA sequencing to contain thefull-length thrA gene having the correct sequence (SEQ ID NO: 88). Thecorresponding protein is represented by SEQ ID NO: 89.

An aspartate kinase/homoserine dehydrogenase with strongly decreasedsensitivity for inhibition by threonine was constructed by site directedmutagenesis, replacing serine in position 345 with phenylalanine(S345F). Site-directed mutagenesis was carried out using the direct andreverse primers 5′-TGTCTCGAGCCCGTATTTTCGTGGTGCTG-3′ (SEQ ID NO: 90) and5′-CAGCACCACGAAAATACGGGCTCGAGACA-3′ (SEQ ID NO: 91) and the pET28-thrAwtplasmid as the template. A single point mutation to change the aminoacid sequence was introduced by PCR (Phusion 1U, HF buffer 20% (v/v),dNTPs 0.2 mM, direct and reverse primers 0.04 μM each, template plasmid30-50 ng, water). Plasmids created by PCR contained a new restrictionsite for XhoI (underlined) introduced by silent mutation in addition tothe functional mutation to facilitate identification of mutated clones.The PCR products were digested by DpnI at 37° C. for 1 h to removetemplate DNA, and transformed into DH5a competent E. coli cells (NEB).The mutated plasmid pET_Ec_thrA_S345F was identified by restriction siteanalysis and verified to carry the desired mutation by DNA sequencing.

The thrAS345F coding region of the bifunctional E. coli aspartatekinase/homoserine dehydrogenase was obtained by PCR using the plasmidpET_Ec_thrA_S345F as the template (SEQ ID NO: 92). The whole codingregion was amplified by PCR using high fidelity polymerase Phusion™(Finnzymes) and the direct and reverse primers5′-TATAATGAGCTCGTTTAACTTTAAGAAGGAGATATACCATGCGAGTGTTGA AGTTCGGCG-3′ (SEQID NO: 93) and 5′-TATAATCCCGGGTCAGACTCCTAACTTCCA-3′ (SEQ ID NO: 94) thatintroduced a SacI and XmaI restriction site (underlined) upstream of thestart codon and downstream of the stop codon, respectively. The directprimer includes the ribosome binding site (bold face) sequence of pET28.The PCR product was digested with SacI and XmaI, ligated into thecorresponding sites of either pEXT20 or pACT3 (Dykxhoorn, St Pierre, &Linn, 1996), using T4 DNA ligase (Biolabs), and transformed into E. coliDH5a cells. The resulting pEXT20-op-HMS2_step1 and pACT3-op-HMS2_step1plasmids were isolated and shown by DNA sequencing to have the correctsequence.

Escherichia coli aspartate semialdehyde dehydrogenase asd was amplifiedby PCR using high fidelity polymerase Phusion™ (Finnzymes) and thedirect and reverse primers5′-TATAATCCCGGGGTTTAACTTTAAGAAGGAGATATACCATGAAAAATGTTG GTTTTATCGGC-3′(SEQ ID NO: 95) and 5′-TATAATGGATCCTTACGCCAGTTGACGAAG-3′ (SEQ ID NO: 96)that introduced a XmaI and BamHI restriction site upstream of the startcodon and downstream of the stop codon, respectively (SEQ ID NO: 98).The direct primer includes the ribosome binding site sequence of pET28.Genomic DNA of E coli MG1655 was used as the template. The PCR productwas digested with XmaI and BamHI, ligated into the corresponding sitesof pEXT20-op-HMS2_step1 and pACT3-op-HMS2_step1, directly downstream theE. coli thrA gene, using T4 DNA ligase (Biolabs), and transformed intoE. coli DH5a cells. The resulting pEXT20-op-HMS2 and pACT3-op-HMS2plasmids were isolated and shown by DNA sequencing to have the correctsequence.

Example 6 Construction of Plasmids for Overexpression ofPhosphoenolpyruvate (PEP) Carboxykinase, PEP Carboxylase, PyruvateKinase, Pyruvate Carboxylase, Isocitrate Lyase Enzymes and the GalactoseSymporter Permease

The plasmid pACT3-pck harbouring the PEP carboxykinase encoding pck geneof E. coli was constructed by amplifying the pck coding sequence usinggenomic DNA from E. coli MG1655 as the template and the forward andreverse primers, respectively, 5′ TATAATCCCGGGATGCGCGTTAACAATGGTTTGACC3′(SEQ ID NO: 119) and 5′ TATAATTCTAGATTACAGTTTCGGACCAGCCG3′ (SEQ ID NO:120). The DNA fragment was digested with XmaI and XbaI, ligated into thecorresponding sites of the pACT3 expression vector (Dykxhoorn et al.,1996) using T4 DNA ligase (Biolabs), and transformed into E. coli DH5acells. The transformants were selected on solid LB medium containingchloramphenicol (25 μg/mL). The resulting plasmid was isolated andcorrect insertion of the pck gene was verified by sequencing. PlasmidspACT3-aceA, pACT3-ppc, pACT3-galP, pACT3-pck and pACT3-pycA harbouring,respectively, aceA, ppc, galP, or pck (all E. coli) or pycA fromLactococcus lactis were constructed analogously using the primers listedin Table 9.

TABLE 9 Primers used for construction ofplasmids for gene overexpression.Restriction sites used for cloning into pACT3 are underlined Gene PrimerLinker Sequence Ec_pck Ec_pck_clon_for XmaI tataatcccgggatgcgcgttaacaatggttt gacc (SEQ ID No. 121) Ec_pck_clon_rev XbaItataattctagattac agtttcggaccagccg (SEQ ID No. 122) Ec_ppcEc_ppc_clon_for XmaI tataatcccgggatga acgaacaatattcc (SEQ ID No. 123)Ec_ppc_clon_rev XbaI tataattctagattag ccggtattacgcat (SEQ ID No. 124)Ec_aceA Ec_aceA_clon_for XmaI tataatcccgggatga aaacccgtacacaaca aatt(SEQ ID No. 125) Ec_aceA_clon_rev XbaI tataattctagattag aactgcgattcttcag(SEQ ID No. 126) Ll_pycA Ll_pycA_clon_for XmaI tataatcccgggatgaaaaaactactcgtcgc caat (SEQ ID No. 127) Ll_pycA_clon_rev XbaItataattctagattaa ttaatttcgattaaca (SEQ ID No. 128) Ec_galPEc_galP_clon_for XmaI tataatcccgggatgc ctgacgctaaaaaaca ggggcggt(SEQ ID No. 129) Ec_galP_clon_rev XbaI tataattctagattaa tcgtgagcgcctatttc (SEQ ID No. 130)

Example 7 Construction of the Plasmid for Overexpression of theHomoserine Transaminase and the OHB Reductase

The coding sequence of the branched chain amino transferase, IlvE, fromE. coli was PCR amplified using the forward and reverse primers5′-ACAATTTCACACAGGAAACAGAATTCGAGCTCGGTACCGTTTAACTTTAAGAAGGAGATATACCATGACCACGAAGAAAGCTGATTAC-3′ (SEQ ID NO: 131) and5′-GGATAACTTTTTTACGTTGTTTATCAGCCATGGTATATCTCCTTCTTAAAGTTAAACGGATCCTTATTGATTAACTTG-3′ (SEQ ID NO: 132), respectively, andplasmid pET28-Ec-ilvE (Example 4) as the template. The coding sequenceof lactate dehydrogenase, LdhA, from L. lactis was PCR amplified usingthe forward and reverse primers5′-TAATATGGATCCGTTTAACTTTAAGAAGGAGATATACCATGGCTGATAAACAACGTAAAAAAGTTATCC-3′ (SEQ ID NO: 133) and5′-CAATGCGGAATATTGTTCGTTCATGGTATATCTCCTTCTTAAAGTTAAACTCTAGATTAGTTTTTAACTGCAGAAGCAAATTC-3′ (SEQ ID NO: 134), respectively, andplasmid pET28-Ll-ldhA (Example 1) as the template. The amplified PCRfragments were fused in an overlap extension PCR by adding 150 ng ofeach fragment to 50 μL of the reaction mix and running a PCR usingprimers 5′-ACAATTTCACACAGGAAACAGAATTCGAGCTCGGTACCGTTTAACTTTAAGAAGGAGATATACCATGACCACGAAGAAAGCTGATTAC-3′ (SEQ ID NO: 135) and5′-CAATGCGGAATATTGTTCGTTCATGGTATATCTCCTTCTTAAAGTTAAACTCTAGATTAGTTTTTAACTGCAGAAGCAAATTC-3′ (SEQ ID NO: 136). The resulting PCRfragment was purified, digested with Kpnl and XbaI, and ligated into thecorresponding sites of pEXT20 (Dykxhoorn, St Pierre, & Linn, 1996) usingT4 DNA ligase (Fermentas). The ligation product was transformed into E.coli DH5a. The resulting plasmid pEXT20-DHB was isolated and shown byDNA sequencing to contain the correct full-length coding sequences ofEc-ilvE and Ll-ldhA. The plasmid was then transformed into E. coliMG1655-derived mutant strains and tested regarding DHB production.

Example 8 Construction of Optimized Strains for DHB Production

Several genes were disrupted in E. coli strain MG1655 in order tooptimise carbon flux repartitioning and cofactor supply for DHBproduction. Gene deletions were carried out using phage transductionmethod, or the lambda red recombinase method according to Datsenko etal. (Datsenko & Wanner, 2000).

Protocol for Introduction of Gene Deletions Using the Phage TransductionMethod:

Strains carrying the desired single deletions were obtained from theKeio collection (Baba et al., 2006). Phage lysates of single deletionmutants were prepared by inoculating 10 mL of LB medium containing 50μg/mL kanamycin, 2 g/L glucose, and 5 mM CaCl₂ with 100 μL of overnightprecultures. Following an incubation of 1 h at 37° C., 200 μL of phagelysate prepared from the wild-type MG1655 strain were added, andcultures were incubated for another 2-3 h until cell lysis hadcompleted. After addition of 200 μL chloroform, cell preparations werefirst vigorously vortexted and then centrifuged for 10 min at 4500×g.The clear lysate was recovered and stored at 4° C.

The receptor strain was prepared for phage transduction by an overnightcultivation at 37° C. in LB medium. A volume of 1.5 mL of the preculturewas centrifuged at 1500×g for 10 min. The supernatant was discarded andthe cell pellet was resuspended in 600 μl of a solution containing 10 mMMgSO₄ and 5 mM CaCl₂. The transduction was carried out by mixing 100 μLof the solution containing the receptor strain with 100 μL of lysate andincubating this mixture at 30° C. for 30 min. Thereafter, 100 μL of a 1Msodium citrate solution were added followed by vigorous vortexing. Afteraddition of 1 mL LB medium, the cell suspension was incubated at 37° C.for 1 h before spreading the cells on LB agar dishes containing 50 μg/mLkanamycin. Clones able to grow in presence of the antibiotic wereconfirmed by colony PCR to contain the desired deletion using theprimers listed in Table 11. After the introduction of each genedeletion, the antibiotic marker was removed as described above followingthe method of (Cherepanov & Wackernagel, 1995). The deletions ΔldhA,ΔadhE, ΔmetA, ΔthrB, ΔrhtB, and ΔlldD were successively introduced bythe described method.

Protocol for Introduction of Gene Deletions Using the Lambda-RedRecombinase Method:

The deletion cassettes were prepared by PCR using high fidelitypolymerase Phusion™ (Finnzymes), and the FRT-flanked kanamycinresistance gene (kan) of plasmid pKD4 as the template (Datsenko &Wanner, 2000). Sense primers contained sequences corresponding to the 5′end of each targeted gene (underlined) followed by 20 bp correspondingto the FRT-kan-FRT cassette of pKD4. Anti-sense primers containedsequences corresponding to the 3′ end region of each targeted gene(underlined) followed by 20 bp corresponding to the cassette. Theprimers are described in Table 10. PCR products were digested with DpnIand purified prior to transformation.

E. coli MG1655 strain was rendered electro-competent by growing thecells to an OD₆₀₀ of 0.6 in LB liquid medium at 37° C., concentratingthe cells 100-fold, and washing them twice with ice-cold 10% glycerol.The cells were transformed with plasmid pKD46 (Datsenko & Wanner, 2000)by electroporation (2.5 kV, 200 Ω, 25 μF, in 2 mm gap cuvettes).Transformants were selected at 30° C. on ampicillin (100 μg/mL) LB solidmedium.

Disruption cassettes were transformed into electro-competent E. colistrains harbouring the lambda Red recombinase-expressing plasmid pKD46.The cells were grown at 30° C. in liquid SOB medium containingampicillin (100 μg/mL). The lambda red recombinase system was induced byadding 10 mM arabinose when OD₆₀₀ of the cultures reached 0.1. Cellswere further grown to an OD₆₀₀ of 0.6 before they were harvested bycentrifugation, washed twice with ice-cold 10% glycerol, and transformedwith the disruption cassette by electroporation. After an overnightphenotypic expression at 30° C. in LB liquid medium, cells were platedon solid LB medium containing 25 μg/mL kanamycin. Transformants wereselected after cultivation at 30° C.

The gene replacement was verified by colony PCR using Crimson Taqpolymerase (NEB). A first reaction was carried out with the flankinglocus-specific primers (see Table 11) to verify simultaneous loss of theparental fragment and gain of the new mutant specific fragment. Twoadditional reactions were done by using one locus-specific primertogether with one of the corresponding primers k1rev, or k2 for (seeTable 11) that align within the FRT-kanamycin resistance cassette (senselocus primer/k1rev and k2for/reverse locus primer).

The resistance gene (FRT-kan-FRT) was subsequently excised from thechromosome using the FLP recombinase-harbouring plasmid pCP20(Cherepanov & Wackernagel, 1995) leaving a scar region containing oneFRT site. pCP20 is an ampicillin and CmR plasmid that showstemperature-sensitive replication and thermal induction of FLPrecombinase synthesis. Kanamycin resistant mutants were transformed withpCP20, and ampicillin-resistant transformants were selected at 30° C.Transformants were then grown on solid LB medium at 37° C. and testedfor loss of all antibiotic resistances. Excision of the FRT-kanamycincassette was analysed by colony PCR using crimson taq polymerase and theflanking locus-specific primers (Table 11). Multiple deletions wereobtained by repeating the above described steps.

TABLE 10 Primers used for gene disruptions.Sequences homologous to target genes are underlined Gene Primer SequenceldhA Δ_ldhA_for gaaggttgcgcctacactaagcatagttgttgatgagtgtaggctggagctgcttc(SEQ ID No. 137) Δ_ldhA_revttaaaccagttcgttcgggcaggtttcgcctttttcatgggaattagccatggtcc SEQ ID No. 138)adhE Δ_adhE_foratggctgttactaatgtcgctgaacttaacgcactcgtagagcgtgtgtaggctggagctgcttc(SEQ ID No. 139) Δ_adhE_revttaagcggattttttcgcttttttctcagctttagccggagcagccatatgaatatcctccttag(SEQ ID No. 140) ackA Δ_ackA_foratgtcgagtaagttagtactggttctgaactgcggtagttcttcagtgtaggctggagctgcttc(SEQ ID No. 141 Δ_ackA_revtcaggcagtcaggcggctcgcgtcttgcgcgataaccagttcttccatatgaatatcctccttag(SEQ ID No. 142) focA- Δ_focA-pflB_forttactccgtatttgcataaaaaccatgcgagttacgggcctataagtgtaggctggagctgcttc pflB(SEQ ID No. 143) Δ_focA-pflB_revatagattgagtgaaggtacgagtaataacgtcctgctgctgttctcatatgaatatcctccttag(SEQ ID No. 144) pta Δ_pta_forgtgtcccgtattattatgctgatccctaccggaaccagcgtcggtgtgtaggctggagctgcttc(SEQ ID No. 145) Δ_pta_revttactgctgctgtgcagactgaatcgcagtcagcgcgatggtgtacatatgaatatcctccttag(SEQ ID No. 146) poxB Δ_poxB_foratgaaacaaacggttgcagcttatatcgccaaaacactcgaatcggtgtaggctggagctgcttc(SEQ ID No. 147) Δ_poxB_revttaccttagccagtttgttttcgccagttcgatcacttcatcacccatatgaatatcctccttag(SEQ ID No. 148) sad Δ_sad_foratgaccattactccggcaactcatgcaatttcgataaatcctgccgtgtaggctggagctgcttc(SEQ ID No. 149) Δ_sad_revtcagatccggtctttccacaccgtctggatattacagaattcgtgcatatgaatatcctccttag(SEQ ID No. 150) gabD Δ_gabD_foratgaaacttaacgacagtaacttattccgccagcaggcgttgattgtgtaggctggagctgcttc(SEQ ID No. 151) Δ_gabD_revttaaagaccgatgcacatatatttgatttctaagtaatcttcgatcatatgaatatcctccttag(SEQ ID No. 152) gadA Δ_gadA_foratggaccagaagctgttaacggatttccgctcagaactactcgatgtgtaggctggagctgcttc(SEQ ID No. 153) Δ_gadA_revtcaggtgtgtttaaagctgttctgctgggcaataccctgcagtttcatatgaatatcctccttag(SEQ ID No. 154) gadB Δ_gadB_foratggataagaagcaagtaacggatttaaggtcggaactactcgatgtgtaggctggagctgcttc(SEQ ID No. 155) Δ_gadB_revtcaggtatgtttaaagctgttctgttgggcaataccctgcagtttcatatgaatatcctccttag(SEQ ID No. 156) gadC Δ_gadC_foratggctacatcagtacagacaggtaaagctaagcagctcacattagtgtaggctggagctgcttc(SEQ ID No. 157) Δ_gadC_revttagtgtttcttgtcattcatcacaatatagtgtggtgaacgtgccatatgaatatcctccttag(SEQ ID No. 158) sfcA Δ_sfcA_foratggaaccaaaaacaaaaaaacagcgttcgctttatatcccttacgtgtaggctggagctgcttc(SEQ ID No. 159) Δ_sfcA_revttagatggaggtacggcggtagtcgcggtattcggcttgccagaacatatgaatatcctccttag(SEQ ID No. 160) maeB Δ_maeB_foratggatgaccagttaaaacaaagtgcacttgatttccatgaatttgtgtaggctggagctgcttc(SEQ ID No. 161) Δ_maeB_revttacagcggttgggtttgcgcttctaccacggccagcgccaccatcatatgaatatcctccttag(SEQ ID No. 162) ppc Δ_ppc_foratgaacgaacaatattccgcattgcgtagtaatgtcagtatgctcgtgtaggctggagctgcttc(SEQ ID No. 163) Δ_ppc_revttagccggtattacgcatacctgccgcaatcccggcaatagtgaccatatgaatatcctccttag(SEQ ID No. 164) pykA Δ_pykA_foratgtccagaaggcttcgcagaacaaaaatcgttaccacgttaggcgtgtaggctggagctgcttc(SEQ ID No. 165) Δ_pykA_revttactctaccgttaaaatacgcgtggtattagtagaacccacggtcatatgaatatcctccttag(SEQ ID No. 166) pykF Δ_pykF_foratgaaaaagaccaaaattgtttgcaccatcggaccgaaaaccgaagtgtaggctggagctgcttc(SEQ ID No. 167) Δ_pykF_revttacaggacgtgaacagatgcggtgttagtagtgccgctcggtaccatatgaatatcctccttag(SEQ ID No. 168) mgsA Δ_mgsA_foratggaactgacgactcgcactttacctgcgcggaaacatattgcggtgtaggctggagctgcttc(SEQ ID No. 169) Δ_mgsA_revttacttcagacggtccgcgagataacgctgataatcggggatcagcatatgaatatcctccttag(SEQ ID No. 170) iclR Δ_iclR_foratggtcgcacccattcccgcgaaacgcggcagaaaacccgccgttgtgtaggctggagctgcttc(SEQ ID No. 171) Δ_iclR_revtcagcgcattccaccgtacgccagcgtcacttccttcgccgctttcatatgaatatcctccttag(SEQ ID No. 172) icd Δ_icd_foratggaaagtaaagtagttgttccggcacaaggcaagaagatcaccgtgtaggctggagctgcttc(SEQ ID No. 173) Δ_icd_revttacatgttttcgatgatcgcgtcaccaaactctgaacatttcagcatatgaatatcctccttag(SEQ ID No. 174) sucA Δ_sucA_foratgcagaacagcgctttgaaagcctggttggactcttcttacctcgtgtaggctggagctgcttc(SEQ ID No. 175) Δ_sucA_revttattcgacgttcagcgcgtcattaaccagatcttgttgctgtttcatatgaatatcctccttag(SEQ ID No. 176) sucB Δ_sucB_foratgagtagcgtagatattctggtccctgacctgcctgaatccgtagtgtaggctggagctgcttc(SEQ ID No. 177) Δ_sucB_revctacacgtccagcagcagacgcgtcggatcttccagcaactctttcatatgaatatcctccttag(SEQ ID No. 178) frdA Δ_frdA_forgtgcaaacctttcaagccgatcttgccattgtaggcgccggtggcgtgtaggctggagctgcttc(SEQ ID No. 179) Δ_frdA_revtcagccattcgccttctccttcttattggctgcttccgccttatccatatgaatatcctccttag(SEQ ID No. 180) frdB Δ_frdB_foratggctgagatgaaaaacctgaaaattgaggtggtgcgctataacgtgtaggctggagctgcttc(SEQ ID No. 181) Δ_frdB_revttagcgtggtttcagggtcgcgataagaaagtctttcgaactttccatatgaatatcctccttag(SEQ ID No. 182) frdC Δ_frdC_foratgacgactaaacgtaaaccgtatgtacggccaatgacgtccaccgtgtaggctggagctgcttc(SEQ ID No. 183) Δ_frdC_revttaccagtacagggcaacaaacaggattacgatggtggcaaccaccatatgaatatcctccttag(SEQ ID No. 184) frdD Δ_frdD_foratgattaatccaaatccaaagcgttctgacgaaccggtattctgggtgtaggctggagctgcttc(SEQ ID No. 185) Δ_frdD_revttagattgtaacgacaccaatcagcgtgacaactgtcaggatagccatatgaatatcctccttag(SEQ ID No. 186) ptsI Δ_ptsI_foratgatttcaggcattttagcatccccgggtatcgctttcggtaaagtgtaggctggagctgcttc(SEQ ID No. 187) Δ_ptsI_revttagcagattgttttttcttcaatgaacttgttaaccagcgtcatcatatgaatatcctccttag(SEQ ID No. 188) ptsG Δ_ptsG_foratgtttaagaatgcatttgctaacctgcaaaaggtcggtaaatcggtgtaggctggagctgcttc(SEQ ID No. 189) Δ_ptsG_revttagtggttacggatgtactcatccatctcggttttcaggttatccatatgaatatcctccttag(SEQ ID No. 190) lacI Δ_lacI_forgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtcgtgtaggctggagctgcttc(SEQ ID No. 191) Δ_lacI_revtcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatcatatgaatatcctccttag(SEQ ID No. 192) lldD Δ_lldD_foratgattatttccgcagccagcgattatcgcgccgcagcgcaacgcgtgtaggctggagctgcttc(SEQ ID No. 193) Δ_lldD_revctatgccgcattccctttcgccatgggagccagtgccgcaggcaacatatgaatatcctccttag(SEQ ID No. 194) pgi Δ_pgi_foratgaaaaacatcaatccaacgcagaccgctgcctggcaggcactagtgtaggctggagctgcttc(SEQ ID No. 195) Δ_pgi_revttaaccgcgccacgctttatagcggttaatcagaccattggtcgacatatgaatatcctccttag(SEQ ID No. 196) metA Δ_metA_foratgccgattcgtgtgccggacgagctacccgccgtcaatttcttggtgtaggctggagctgcttc(SEQ ID No. 197) Δ_metA_revttaatccagcgttggattcatgtgccgtagatcgtatggcgtgatcatatgaatatcctccttag(SEQ ID No. 198) thrB Δ_thrB_foratggttaaagtttatgccccggcttccagtgccaatatgagcgtcgtgtaggctggagctgcttc(SEQ ID No. 199) Δ_thrB_revttagttttccagtactcgtgcgcccgccgtatccagccggcaaatcatatgaatatcctccttag(SEQ ID No. 200) lysA Δ_lysA_foratgccacattcactgttcagcaccgataccgatctcaccgccgaagtgtaggctggagctgcttc(SEQ ID No. 201) Δ_lysA_revttaaagcaattccagcgccagtaattcttcgatggtctggcgacgcatatgaatatcctccttag(SEQ ID No. 202) eda Δ_eda_foratgaaaaactggaaaacaagtgcagaatcaatcctgaccaccggcgtgtaggctggagctgcttc(SEQ ID No. 203) Δ_eda_revctcgatcgggcattttgacttttacagcttagcgccttctacagccatatgaatatcctccttag(SEQ ID No. 204) recA Δ_recA_foratggctatcgacgaaaacaaacagaaagcgttggcggcagcactggtgtaggctggagctgcttc(SEQ ID No. 205) Δ_recA_revttaaaaatcttcgttagtttctgctacgccttcgctatcatctaccatatgaatatcctccttag(SEQ ID No. 206) asd Δ_asd_foratgaaaaatgttggttttatcggctggcgcggtatggtcggctccgtgtaggctggagctgcttc(SEQ ID No. 207) Δ_asd_revttacgccagttgacgaagcatccgacgcagcggctccgcggcccccatatgaatatcctccttag(SEQ ID No. 208)

TABLE 11 Primer pairs used for verification of gene disruptions DeletedSequence (5′ - 3′) gene Forwardprimer Reverse primer K2 for/cggtgccctgaatgaactgc cagtcatagccgaatagcct k1 rev (SEQ ID No. 209)(SEQ ID No. 210) ldhA atacgtgtcccgagcggtag tacacatcccgccatcagca(SEQ ID No. 211) (SEQ ID No. 212) adhE GaagtaaacgggaaaatcaaAgaagtggcataagaaaacg (SEQ ID No. 213) (SEQ ID No. 214) ackAccattggctgaaaattacgc gttccattgcacggatcacg (SEQ ID No. 215)(SEQ ID No. 216) focA_pflB atgccgtagaagccgccagt tgttggtgcgcagctcgaag(SEQ ID No. 217) (SEQ ID No. 218) pta gcaaatctggtttcatcaactcccttgcacaaaacaaagt (SEQ ID No. 219) (SEQ ID No. 220) poxBggatttggttctcgcataat agcattaacggtagggtcgt (SEQ ID No. 221)(SEQ ID No. 222) sad gctgattctcgcgaataaac aaaaacgttcttgcgcgtct(SEQ ID No. 223) (SEQ ID No. 224) gabD tctgtttgtcaccaccccgcAagccagcacctggaagcag (SEQ ID No. 225) (SEQ ID No. 226) gadAaagagctgccgcaggaggat gccgccctcttaagtcaaat (SEQ ID No. 227)(SEQ ID No. 228) gadB ggattttagcaatattcgct cctaatagcaggaagaagac(SEQ ID No. 229) (SEQ ID No. 230) gadC gctgaactgttgctggaagaggcgtgcttttacaactaca (SEQ ID No. 231) (SEQ ID No. 232) sfcAtagtaaataacccaaccggc tcagtgagcgcagtgtttta (SEQ ID No. 233)(SEQ ID No. 234) maeB attaatggtgagagtttgga tgcttttttttattattcgc(SEQ ID No. 235) (SEQ ID No. 236) ppc gctttataaaagacgacgaagtaacgacaattccttaagg (SEQ ID No. 237) (SEQ ID No. 238) pykAtttatatgcccatggtttct atctgttagaggcggatgat (SEQ ID No. 239)(SEQ ID No. 240) pykF ctggaacgttaaatctttga ccagtttagtagctttcatt(SEQ ID No. 241) (SEQ ID No. 242) iclR gatttgttcaacattaactcatcggtgcgattaacagacaccctt (SEQ ID No. 243) (SEQ ID No. 244) mgsAtctcaggtgctcacagaaca tatggaagaggcgctactgc (SEQ ID No. 245)(SEQ ID No. 246) icd cgacctgctgcataaacacc tgaacgctaaggtgattgca(SEQ ID No. 247) (SEQ ID No. 248) sucA acgtagacaagagctcgcaacatcacgtacgactgcgtcg (SEQ ID No. 249) (SEQ ID No. 250) sucBtgcaactttgtgctgagcaa tatcgcttccgggcattgtc (SEQ ID No. 251)(SEQ ID No. 252) frdA Aaatcgatctcgtcaaatttcagac aggaaccacaaatcgccata(SEQ ID No. 253) (SEQ ID No. 254) frdB gacgtgaagattactacgctagttcaatgctgaaccacac (SEQ ID No. 255) (SEQ ID No. 256) frdCtagccgcgaccacggtaagaaggag cagcgcatcacccggaaaca (SEQ ID No. 257)(SEQ ID No. 258) frdD atcgtgatcattaacctgat ttaccctgataaattaccgc(SEQ ID No. 259) (SEQ ID No. 260) ptsG ccatccgttgaatgagtttttggtgttaactggcaaaatc (SEQ ID No. 261) (SEQ ID No. 262) ptsIgtgacttccaacggcaaaag ccgttggtttgatagcaata (SEQ ID No. 263)(SEQ ID No. 264) lacI Gaatctggtgtatatggcga Tcttcgctattacgccagct(SEQ ID No. 265) (SEQ ID No. 266) lldD CgtcagcggatgtatctggtGcggaatttctggttcgtaa (SEQ ID No. 267) (SEQ ID No. 268) pgiTtgtcaacgatggggtcatg Aaaaatgccgacataacgtc (SEQ ID No. 269)(SEQ ID No. 270) lysA Tctcaaagcgcgcaagttcg Ggtattgatgtaccgggtgagatt(SEQ ID No. 271) (SEQ ID No. 272) metA TcgacagaacgacaccaaatCactgtgaacgaaggatcgt (SEQ ID No. 273) (SEQ ID No. 274) thrBTgttggcaatattgatgaag Gacatcgctttcaacattgg (SEQ ID No. 275)(SEQ ID No. 276) eda Gacagacaggcgaactgacg Gcgcagatttgcagattcgt(SEQ ID No. 277) (SEQ ID No. 278) recA TggcggcagtgaagagaagcGcaataacgcgctcgtaatc (SEQ ID No. 279) (SEQ ID No. 280) asdAcaaagcaggataagtcgca Gacttcaggtaaggctgtga (SEQ ID No. 281)(SEQ ID No. 282) rhtA CAGAGAACTGCGTAAGTATTACGCATAGTGGTAACAAGCGTGAAAAACAA (SEQ ID No. 283) (SEQ ID No. 284) rhtBATGAAGACTCCGTAAACGTTTCCCC CAAAAATAGACACACCGGGAGTTCA (SEQ ID No. 285)(SEQ ID No. 286)

The plasmid co-expressing aspartate kinase, aspartate semialdehydedehydrogenase, and homoserine dehydrogenase (pACT3-op-HMS1) wastransformed together with the plasmid expressing the homoserinetransaminase and the OHB reductase (pEXT20-DHB) into the optimized hoststrains. Transformants were selected on solid LB medium containingchloramphenicol (25 μg/mL) and ampicillin (100 μg/mL). Non-exclusiveexamples of constructed strains are listed in Table 12.

TABLE 12 Examples of strains constructed for DHB production StrainRelevant Genotype MG1655 Wild-type ECE73 ΔldhA ΔadhE ΔmetA ΔthrB ECE74ΔldhA ΔadhE ΔmetA ΔthrB pACT3-op-HMS1 ECE75 ΔldhA ΔadhE ΔmetA ΔthrBpEXT20-DHB ECE76 ΔldhA ΔadhE ΔmetA ΔthrB pACT3-op-HMS1 pEXT20-DHB ECE77ΔldhA ΔadhE ΔmetA ΔthrB ΔlldD pACT3-op-HMS1 pEXT20-DHB ECE78 ΔldhA ΔadhEΔmetA ΔthrB ΔrhtB pACT3-op-HMS1 pEXT20-DHB

It is understood that removal of the lacI gene from the backbone of theabove described plasmids along with the genomic deletion of lacI in thehost strain may render protein expression from above described plasmidsconstitutive.

Example 9 Demonstration of the Zymotic Production of DHB Via theHomoserine-OHB Pathway

Strains and cultivation conditions: Experiments were carried out withstrains listed in Table 12. All cultivations were carried out at 37° C.on an Infors rotary shaker running at 170 rpm. Overnight cultures (3 mLmedium in test tube) were inoculated from glycerol stocks and used toadjust an initial OD₆₀₀ of 0.05 in 100 mL growth cultures cultivated in500 mL shake flasks. IPTG was added at a concentration of 1 mmol/L whenOD₆₀₀ in the growth cultures reached 0.8. One liter culture mediumcontained, 20 g glucose, 18 g Na₂HPO₄*12 H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 2g NH₄Cl, 0.5 g MgSO₄*7 H₂O, 0.015 CaCl₂*2 H₂O, 1 mL of 0.06 mol/L FeCl₃stock solution prepared in 100 times diluted concentrated HCl, 2 mL of10 mM thiamine HCl stock solution, 20 g MOPS and 1 mL of trace elementsolution (containing per liter: 0.04 g Na₂EDTA*2H₂O, 0.18 g CoCl₂*6 H₂O,ZnSO4*7 H₂O, 0.04 g Na₂MoO4*2 H₂O, 0.01 g H₃BO₃, 0.12 g MnSO₄*H₂O, 0.12g CuCl₂*H2O.). Medium pH was adjusted to 7 and medium wasfilter-sterilized. The antibiotics kanamycin sulphate, ampicillin, andchloramphenicol were added at concentrations of 50 mg/L, 100 mg/L, and25 mg/L, respectively, when necessary.

Estimation of DHB concentration by LC-MS analyses: Liquid anion exchangechromatography was performed on an ICS-3000 system from Dionex(Sunnyvale, USA) equipped with an automatic eluent (KOH) generatorsystem (RFIC, Dionex), and an autosampler (AS50, Dionex) holding thesamples at 4° C. Analytes were separated on an lonPac AS11 HC (250×2 mm,Dionex) column protected by an AG11 HC (50×2 mm, Dionex) pre-column.Column temperature was held at 25° C., flow rate was fixed at 0.25mL/min, and analytes were eluted applying the KOH gradient describedearlier (Groussac E, Ortiz M & Francois J (2000): Improved protocols forquantitative determination of metabolites from biological samples usinghigh performance ionic-exchange chromatography with conductimetric andpulsed amperometric detection. Enzyme. Microb. Technol. 26, 715-723).Injected sample volume was 15 μL. For background reduction, an ASRSultra II (2 mm, external water mode, 75 mA) anion suppressor was used.Analytes were quantified using a mass-sensitive detector (MSQ Plus,Thermo) running in ESI mode (split was ⅓, nitrogen pressure was 90 psi,capillary voltage was 3.5 kV, probe temperature was 450° C.).

Results:

After 24 h cultivation, the DHB concentration in the supernatant ofdifferent strains was quantified by LC-MS analyses. The strains ECE73,ECE74, ECE75, and ECE76 had produced 0 mg/L, 3.7 mg/L, 0.67 mg/L, and11.9 mg/L of DHB, respectively.

REFERENCES

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The invention claimed is:
 1. A method for the preparation of2,4-dihydroxybutyrate (2,4-DHB) from homoserine comprising: deaminatinghomoserine to form 2-oxo-4-hydroxybutyrate (OHB), where the deaminationof homoserine is catalyzed by an enzyme having homoserine transaminaseactivity, wherein the enzyme having homoserine transaminase activity isproduced via a transformed host microorganism that comprises a firstchimeric gene including a first nucleic acid sequence encoding theenzyme having homoserine transaminase activity for converting theprimary amino group of homoserine to a carbonyl group to obtain OHB, andthe enzyme having homoserine transaminase activity is selected from thegroup consisting of aspC from Escherichia coli (SEQ ID NO: 64), ilvEfrom Escherichia coli (SEQ ID NO: 60), bcaT from Lactococcus lactis (SEQID NO: 68), tyrB from Escherichia coli (SEQ ID NO: 62), araT fromLactococcus lactis (SEQ ID NO: 66), and ARO8 from Saccharomycescerevisiae (SEQ ID NO: 70), or is selected from any sequence sharing asequence identity of at least 90% with at least one of the sequences ofsaid enzymes having homoserine transaminase activity; and reducing theOHB to form 2,4-DHB, where the reduction of OHB is catalyzed by anenzyme having OHB reductase activity, wherein the enzyme having OHBreductase activity is produced via the transformed host microorganism,which further comprises a second chimeric gene including a secondnucleic acid sequence encoding the enzyme having OHB reductase activityfor reducing OHB to 2,4-DHB, and the enzyme having OHB reductaseactivity is selected from the group consisting of (L)-malatedehydrogenase from Escherichia coli (SEQ ID NO: 2), (D)-lactatedehydrogenase from Escherichia coli (SEQ ID NO: 4), (L)-lactatedehydrogenase from Lactococcus lactis (SEQ ID NO: 6), (L)-lactatedehydrogenase from Bacillus subtilis (SEQ ID NO: 8), (L)-lactatedehydrogenase from Geobacillus stearothermophilus (SEQ ID NO: 10), thetwo isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus(SEQ ID NO: 12 and SEQ ID NO: 14), or is selected from any sequencesharing a sequence identity of at least 90% with at least one of saidenzymes having OHB reductase activity.
 2. The method of claim 1, whereinthe enzyme having homoserine transaminase activity is selected from thegroup consisting of aspC from Escherichia coli (SEQ ID NO: 64), ilvEfrom Escherichia coli (SEQ ID NO: 60), bcaT from Lactococcus lactis (SEQID NO: 68), tyrB from Escherichia coli (SEQ ID NO: 62), araT fromLactococcus lactis (SEQ ID NO: 66), and ARO8 from Saccharomycescerevisiae (SEQ ID NO: 70), and wherein the enzyme having OHB reductaseactivity is selected from the group consisting of (L)-malatedehydrogenase from Escherichia coli (SEQ ID NO: 2), (D)-lactatedehydrogenase from Escherichia coli (SEQ ID NO: 4), (L)-lactatedehydrogenase from Lactococcus lactis (SEQ ID NO: 6), (L)-lactatedehydrogenase from Bacillus subtilis (SEQ ID NO: 8), (L)-lactatedehydrogenase from Geobacillus stearothermophilus (SEQ ID NO: 10), thetwo isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus(SEQ ID NO: 12 and SEQ ID NO: 14).
 3. The method of claim 1, wherein theenzyme having OHB reductase activity is a lactate dehydrogenasecomprising at least one mutation in position V17, Q85, E89, I226, orA222, said positions being defined by reference to (L)-lactatedehydrogenase from Lactococcus lactis (SEQ. ID NO: 6); or a malatedehydrogenase comprising at least one mutation in position I12, R81,M85, D86, V93, G179, T211, or M227, said positions being defined byreference to (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO:2).
 4. The method of claim 3, wherein the enzyme having OHB reductaseactivity is selected from the group consisting of SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116 and SEQID NO:
 118. 5. A modified microorganism for the preparation of2,4-dihydroxybutyrate (2,4-DHB) from homoserine via a two-step pathwaycomprising: deaminating homoserine to form 2-oxo-4-hydroxybutyrate(OHB), where the deamination of homoserine is catalyzed by an enzymehaving homoserine transaminase activity selected from the groupconsisting of aspC from Escherichia coli (SEQ ID NO: 64), ilvE fromEscherichia coli (SEQ ID NO: 60), bcaT from Lactococcus lactis (SEQ IDNO: 68), tyrB from Escherichia coli (SEQ ID NO: 62), araT fromLactococcus lactis (SEQ ID NO: 66), and ARO8 from Saccharomycescerevisiae (SEQ ID NO: 70), or is selected from any sequence sharing asequence identity of at least 90% with at least one of the sequences ofsaid enzymes having homoserine transaminase activity, and reducing theOHB to form 2,4-DHB, where the reduction of OHB is catalyzed by anenzyme having OHB reductase activity, wherein the enzyme having OHBreductase activity is selected from the group consisting of (L)-malatedehydrogenase from Escherichia coli (SEQ ID NO: 2), (D)-lactatedehydrogenase from Escherichia coli (SEQ ID NO: 4), (L)-lactatedehydrogenase from Lactococcus lactis (SEQ ID NO: 6), (L)-lactatedehydrogenase from Bacillus subtilis (SEQ ID NO: 8), (L)-lactatedehydrogenase from Geobacillus stearothermophilus (SEQ ID NO: 10), andthe two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus(SEQ ID NO: 12 and SEQ ID NO: 14), or is selected from any sequencesharing a sequence identity of at least 90% with at least one of saidenzymes having homoserine transaminase activity; wherein the modifiedmicroorganism is a host microorganism that has been transformed toenhance production of 2,4-DHB compared to a non-transformed hostmicroorganism, the transformed host microorganism comprising: a firstchimeric gene including a first nucleic acid sequence encoding theenzyme having homoserine transaminase activity for converting theprimary amino group of homoserine to a carbonyl group to obtain OHB, anda second chimeric gene including a second nucleic acid sequence encodingthe enzyme having OHB reductase activity for reducing OHB in 2,4-DHB. 6.The modified microorganism of claim 5, wherein the transformed hostmicroorganism has been further transformed to enhance production ofhomoserine compared to the non-transformed host microorganism.
 7. Themodified microorganism of claim 6, wherein the enhanced production ofhomoserine comprises overexpressing one or more additional enzymesselected from the group consisting of aspartate kinase, aspartatesemialdehyde dehydrogenase and homoserine dehydrogenase, wherein theoverexpression of said one or more enzymes is realized by expressing theenzymes from a multicopy plasmid.
 8. The modified microorganism of claim7, wherein the modified microorganism is a bacterium, a yeast, or afungus.
 9. The modified microorganism of claim 7, wherein the expressionof at least of one the enzymatic activities chosen amongphosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase,isocitrate lyase, pyruvate carboxylase, and hexose symporter permease isincreased, and/or at least one of the enzymatic activities chosen amonglactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphateacetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase,2-oxoglutarate dehydrogenase, pyruvate kinase, malic enzyme,phosphoglucose isomerase, phosphoenolpyruvate carboxylase,phosphoenolpyruvate carboxykinase, pyruvate-formate lyase, succinicsemialdehyde dehydrogenase, sugar-transporting phosphotransferase,ketohydroxyglutarate aldolase, homoserine-O-succinyl transferase,homoserine kinase, homoserine efflux transporter, diaminopimelatedecarboxylase, and/or methylglyoxal synthase is decreased.
 10. Themodified microorganism of claim 8, the modified microorganism beingEscherichia coli, which overexpresses at least one of the genes chosenamong ppc (phosphoenol pyruvate carboxylase), pck, aceA, galP, asd,thrA, metL, lysC all E coli; pycA from L lactis, and/or has at least oneof the genes deleted chosen among ldhA, adhE, ackA, pta, poxB, focA,pfIB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG,pgi, fumABCaldA, HdD, iclR, metA, thrB, lysA, eda, rthA, rthB, and rthC.11. The modified microorganism of claim 5, wherein the enzyme havinghomoserine transaminase activity is selected from the group consistingof aspC from Escherichia coli (SEQ ID NO: 64), ilvE from Escherichiacoli (SEQ ID NO: 60), bcaT from Lactococcus lactis (SEQ ID NO: 68), tyrBfrom Escherichia coli (SEQ ID NO: 62), araT from Lactococcus lactis (SEQID NO: 66), and ARO8 from Saccharomyces cerevisiae (SEQ ID NO: 70), andwherein the enzyme having OHB reductase activity is selected from thegroup consisting of (L)-malate dehydrogenase from Escherichia coli (SEQID NO: 2), (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO:4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO: 6),(L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO: 8),(L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ IDNO: 10), and the two isoforms of (L)-lactate dehydrogenase fromOryctalagus cuniculus (SEQ ID NO: 12 and SEQ ID NO: 14).
 12. A method ofproduction of 2,4-DHB comprising the steps of culturing the modifiedmicroorganism of claim 5 in an appropriate culture medium, recovering2,4-DHB from the culture medium.
 13. The method of claim 12 wherein the2,4-DHB is further purified.